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Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK
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Abstract |
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trk2 double deletion mutant in S. pombe. We propose that this pathway and its role in regulating the plasma membrane potential may act as a pleiotropic determinant of sensitivity to chemotherapeutic agents. |
Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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trk2 double mutant displays a more severe cation sensitivity and a greater membrane hyperpolarization than a trk1 mutant, and requires higher levels of potassium in minimal medium to support growth (Ko & Gaber 1991; Ramos et al. 1994; Madrid et al. 1998; Mulet et al. 1999). The potassium transporter, comprised of Trk1 and Trk2 (commonly known as Trk1,2) also regulates and maintains plasma membrane potential through an antagonistic relationship with the Pma1 plasma membrane H+-ATPase. The balance achieved between potassium influx through Trk1,2, and hydrogen efflux via Pma1, sets the steady state of the plasma membrane potential (Serrano 1983, 1989; Perlin et al. 1988; Madrid et al. 1998; Goosens et al. 2000). Loss of Trk1,2 activity causes a build-up of charge at the membrane, affecting transport of other nutrients and resulting in toxic cation uptake into the cell (Vallejo & Serrano 1989; Madrid et al. 1998; Mulet et al. 1999).
Both Trk1,2 and Pma1 are regulated by protein kinases belonging to the Npr1 (nitrogen permease) family. Previous data suggests that Trk1,2 is regulated by the kinases Hal4 (also called Sat4) and Hal5 in response to potassium starvation (Mulet et al. 1999). A hal4hal5 double mutant has a hyperpolarized membrane and is more sensitive to cations than either single mutant, indicating these kinases are partially redundant. Over-expression of either HAL4 or HAL5 cannot rescue the cation sensitivity of a trk1trk2 mutant, but can increase tolerance in wild-type cells, suggesting that Hal4,5 regulate salt tolerance through Trk1,2. Experiments measuring ion transport capability, using Rb+ as a K+ analog, revealed that uptake through Trk1,2 is dependent on Hal4,5 in a gene dosage dependent manner. Thus, both the genetic data and uptake analysis indicate that Hal4,5 positively regulate the Trk1,2 potassium transporter (Madrid et al. 1998; Mulet et al. 1999).
Pma1 is positively regulated by the Ptk2 kinase (Serrano 1983, 1989; McCusker et al. 1987; Vallejo & Serrano 1989; Goosens et al. 2000). PMA1 is an essential gene, but mutants with reduced ATPase activity are resistant to cations and hygromycin B, the opposite phenotype to a trk1trk2 mutant (Perlin et al. 1988; Goosens et al. 2000; Erez & Kahana 2001).
Homologues of Trk1, Trk2 and Pma1 have been identified in fission yeast. SpTrk1 and Trk2 together form the major determinant of potassium uptake (Soldatenkov et al. 1995; Lichtenberg-Frate et al. 1996; Calero et al. 2000). In contrast to the budding yeast homologs, both SpTrk1 and SpTrk2 appear to contribute more equally to potassium transport. Deletion of both trk1+ and trk2+ results in a requirement for potassium, hypersensitivity to sodium ions and hyperpolarization of the plasma membrane (Lichtenberg-Frate et al. 1996; Calero et al. 2000; Calero & Ramos 2003).
In this study, we show that a mutation resulting in sensitivity of fission yeast to a range of chemotherapeutic drugs resides in a gene encoding a protein kinase we have named Hal4. This protein shares sequence homology with Hal4 and Hal5 in S. cerevisiae. We demonstrate that loss of Hal4 function results in hyperpolarization of the plasma membrane. Furthermore, the chemosensitive phenotype is also conserved in S. cerevisiae trk1trk2 and hal4hal5 mutants. In addition to regulation of Trk1,2, our data suggests that Hal4 may regulate other pathway(s) within the cell and that regulation of Trk1,2 by Hal4 is more complex than the simple relationship suggested for the corresponding proteins in S. cerevisiae (Table 1).
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Results |
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A mutagenic screen was set up to generate mutants displaying an acute sensitivity to a particular chemotherapeutic agent. Using this method a mutant was identified which had acquired sensitivity to bleomycin and this was named hal4-1 (Fig. 1A, left hand panels). The mutant displayed an acute sensitivity to a range of stresses, including a number of chemotherapeutic agents other than bleomycin, including doxorubicin, hygromycin B and salts (Fig. 1A, center and right hand panels). Sensitivity was determined as the drug/stress agent concentration sufficient to result in growth inhibition (Table 2). The acute sensitivity to doxorubicin of hal4-1 as compared to wild-type cells appears to be due to increased uptake into the cell whereupon the drug is sequestered in the vacuoles (Fig. 1B).
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mutant. The deletion mutant displayed an identical sensitivity profile to cations, protons and chemotherapeutic agents as hal4-1 indicating that the hal4-1 mutant results in a complete loss of Hal4 function (Fig. 1D). Furthermore, we found that in S. cerevisiae, loss of either HAL4 and HAL5, TRK1, or TRK1 and TRK2 resulted in a similar chemotherapeutic sensitivity profile to hal4-1 (Table 3).
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As shown in Fig. 1A, the hal4-1 mutant displays sensitivity to hygromycin B, which has been shown in previous studies of the Trk1,2 transporter systems in both S. cerevisiae and S. pombe to indicate the presence of a hyperpolarized plasma membrane (Gaber et al. 1988; Mulet et al. 1999; Calero et al. 2000). To test this directly we adapted a method which had previously been used for this purpose in S. cerevisiae, which utilized a potentiometric dye DiSC3(3) (Gaskova et al. 1998, 1999, 2001). If the hal4-1 mutant has a hyperpolarized membrane, this should result in an increased rate of dye uptake because of the cationic nature of the dye. In our experiments we used a confocal microscope to monitor dye uptake. The hal4-1 mutant displays much quicker DiSC3(3) uptake than the wild-type strain (Fig. 2). The first detectable fluorescence in hal4-1 appears after approximately 2 min, whereas in wild-type cells it takes nearly 20 min for the dye to begin to accumulate. The dye binds to unidentified intracellular components, until a saturation point is reached (Gaskova et al. 1998, 1999, 2001). These data show that the hal4-1 mutant has a hyperpolarized plasma membrane compared to wild-type, consistent with a role for Hal4 in regulating membrane potential in fission yeast.
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trk2 is chemosensitive and this sensitivity can be partially rescued by excess KCl
Deletions of both Sptrk1+ and Sptrk2+ were created and combined to give a trk1trk2 double mutant. A trk1trk2 mutant grows slowly on yeast extract and requires the addition of KCl to the media to sustain wild-type growth as was previously reported (Calero et al. 2000). All the cation sensitivities previously observed for the S. cerevisiae trk1trk2 and the hal4hal5 mutants are conserved in our fission yeast trk1trk2 mutant, as has been previously shown (Table 4; Calero et al. 2000). In addition, the S. pombe trk1trk2 double mutant displayed the same pattern of sensitivity to chemotherapeutic agents as both hal4-1 and the S. cerevisiae mutants (Table 4). These findings suggest that Hal4 may regulate the activity of Trk1,2. Interestingly, in the presence of 50 mM KCl as used previously (Calero et al. 2000), we observed a partial rescue of the sensitivity to drugs of both the trk1trk2 and hal4-1 mutants (Fig. 3A). A concentration of 10 mM KCl enables the trk1trk2 mutant to grow at the same rate as wild-type on yeast extract media, but is not sufficient to rescue the sensitivity of this mutant to drugs and cationic stresses (Table 4 and Fig. 3A). However, although excess KCl allowed growth of both hal4-1 and trk1trk2 on lower concentrations of bleomycin, it could only restore some growth to trk1trk2 on higher concentrations (Fig. 3B).
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trk2 mutant does. The addition of an excess of 50 mM KCl to the drug plates, although sufficient to rescue trk1trk2, is insufficient to reverse the hal4-1 sensitivity completely. Addition of higher concentrations of KCl (400 mM) does reverse some of the sensitivities of hal4-1 such that its growth is comparable to that of wild-type cells on plates containing hygromycin B, lithium, sodium, or cadmium. However, for calcium and TMA, 400 mM KCl does not reverse the sensitivity displayed by hal4-1 so that it resembles wild-type growth. The sensitivity of hal4-1 to doxorubicin and bleomycin in the presence of 400 mM KCl displays an intermediate level of reversal, with some sensitivity remaining at higher concentrations (data not shown). This suggests Hal4 may also regulate another pathway or pathways in addition to regulating Trk1,2; and that disruption of these other pathway(s) also results in sensitivity to these stresses, but that this is not dependent on hyperpolarization of the plasma membrane. Hal4 regulation of Trk1,2 is not a simple linear pathway
If the only function of Hal4 is to regulate the activity of Trk1,2 then either a hal41trk1 or hal41trk2 double mutant, or indeed the triple mutant trk1trk2hal4-1 should have exactly the same sensitivity profile as a hal4-1 single mutant. The hal4-1 mutant was crossed to the single mutant strains trk1 and trk2. The tetrads were germinated on yeast extract plates containing excess KCl to ensure that colony growth was not affected by potential reductions in viability. A hal4-1trk2 double mutant is viable, and displays sensitivities equal to but no greater than a hal4-1 mutant alone (data not shown). However, of 22 tetrads, of which there were two parental ditypes, three non-parental ditypes and 17 tetratypes, no colonies corresponding to hal4-1trk1 were seen, suggesting that this double mutant is inviable (data not shown). This suggests that Hal4 does not function in a simple linear pathway with Trk1,2 but may regulate other pathway(s) in the cell, as a trk1trk2 double mutant is viable under these conditions.
Trk1 is a phosphoprotein and becomes hypermodified upon K+ starvation
As the positive regulation of Trk1 and Trk2 by Hal4 and Hal5 in S. cerevisiae, and uptake of ions by Trk1,2 in S. pombe is stimulated by potassium starvation (Ramos & Rodriguez-Navarro 1986; Mulet et al. 1999; Calero et al. 2000), we investigated the effect of potassium starvation on the Trk1,2 transporter in S. pombe. A strain containing a carboxy-terminally HA tagged Trk1 protein was grown in potassium poor mineral media, as previously used (Calero et al. 2000). Trk1 protein runs as a smear of approximately 100 kDa (Fig. 4A, lower arrow). In all immunoprecipitations, lysates containing an HA tagged version of the transcription factor Atf1 was used to ensure that the diffuse nature of the Trk1 band was not due to experimental procedure. In addition, a fraction of each lysate was analyzed by Western blotting for the presence of Mts4 to ensure that equal concentrations of protein extract were used in each immunoprecipitation. After an hour of starvation, a small decrease in mobility of the Trk1 HA protein can be seen (Fig. 4A, upper arrow). This suggests that potassium starvation results in the modification of the Trk1 protein. The modification seems to be transient, as prolonged potassium starvation results in a reversal of this mobility shift.
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To determine whether the modification of Trk1 observed after one hour of potassium starvation was dependent on Hal4, a hal4-1 strain containing HA tagged Trk1 was generated and its response to starvation was compared to that seen in a wild-type background. Surprisingly, the shift in mobility seen after an hour of starvation still occurs in a hal4-1 background and seems to be maintained at 2 h (Fig. 4C, upper arrow). In a wild-type background, this modification appears to have been lost by this point (Fig. 4C, lower arrow). This would suggest that the modification of Trk1 is not due to direct phosphorylation by Hal4, but that Hal4 is required for the regulation of the kinetics of this modification. Furthermore, this modification of Trk1 seems to be a specific response to potassium starvation, as it was not observed after exposure to either bleomycin or sodium stress (data not shown).
In contrast to Trk1, Trk2 does not seem to be phosphorylated in rich media, nor does phosphorylation seem to occur upon potassium starvation (data not shown).
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Discussion |
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In this report we identify a novel kinase, Hal4, which shares sequence homology to Hal4 and Hal5 in S. cerevisiae. Cells lacking Hal4 display acute sensitivity to a range of cations including Li+, Na+, Ca2+, TMA, spermidine and the aminoglycoside antibiotic hygromycin B in an identical pattern to trk1trk2. We show that loss of Hal4 results in hyperpolarization of the plasma membrane, suggesting a conserved function regulating salt homeostasis/membrane potential.
We identified Hal4 through a screen for mutants with acquired sensitivity to the chemotherapeutic drug bleomycin. In addition, the hal4-1 mutant is also sensitive to doxorubicin, daunorubicin, cisplatin and camptothecin. Subsequent analysis of strains defective in Trk1,2 in both S. pombe and S. cerevisiae, and in Hal4 and Hal5 showed this chemosensitivity was conserved across species and thus a consequence of disrupting this pathway. All the chemotherapeutic drugs used, although differing in their molecular targets and gross chemical classes, are cationic. This ionic charge may explain their uptake in cells with hyperpolarized plasma membranes due to defective potassium transport through Trk1,2.
Trk1 is phosphorylated upon potassium starvation. In a hal4-1 mutant, this phosphorylation of Trk1 still occurs, indicating that it is due to another kinase and is not dependent on Hal4. However, phosphorylation is still present after two hours of potassium starvation in a hal4-1 mutant compared to wild-type, suggesting that dephosphorylation is dependent on the activity of Hal4 in some way.
If the only role of Hal4 in the cell was in regulating the Trk1,2 pathway, then one would predict that the hal4-1trk1 double mutant would display the same phenotype as trk1trk2 and therefore be viable. However, we found that the hal4-1trk1 double mutant is not viable which suggests that there is a role for Hal4 in another pathway in addition to the regulation of Trk1 modification in response to potassium starvation.
The hal4-1trk2 mutant is viable and shows no increased sensitivity to cationic stresses than hal4-1 alone. Therefore, it is possible that regulation of Trk1,2 by Hal4 may be mediated through Trk2 and not Trk1. However, Trk2 does not seem to be a phosphoprotein in rich media conditions, and no change in mobility is seen after potassium starvation (data not shown). These data suggest that regulation of Trk1,2 by Hal4 is not mediated by a direct phosphorylation of either Trk1 or Trk2, but instead Hal4 may modulate the kinetics of transporter activation through regulation of an intermediate protein. Furthermore, the genetic interactions indicate that Hal4 also regulates a separate pathway, disruption of which, in conjunction with loss of Trk1, results in a lethal phenotype. This may be completely unrelated to the regulation of salt homeostasis. Current work is therefore focusing on the mechanism of Hal4 regulation of Trk1,2 and on identifying other pathways in which it might play a role.
Whilst this manuscript was in preparation, another study identified Hal4 as a novel Sty1 MAPK interactor (Wang et al. 2005). Their studies show a shared role of these two kinases in determining calcium sensitivity, but distinct roles in regulating ion homeostasis. In the case of Sty1, its role in cation homeostasis is predominantly due to the transcriptional regulation of sod2+, which encodes a Na+/H+ anti-porter. Up-regulation of sod2+ confers resistance to lithium and sodium but not calcium. Hal4 is shown to function through Trk1,2, and Sty1 binding is only required for the response to calcium stress. Their studies are consistent with our own observations on the function of Hal4.
It has previously been suggested that the uptake of toxic cations seen in trk1trk2 and hal4hal5 mutants in S. cerevisiae is due to low affinity transport through other membrane transporters, possibly the result of the cell trying to take up potassium (Goosens et al. 2000). Hyperpolarization of the membrane may affect the activation requirements or substrate specificity of other transporters in the membrane. Attempts to identify transporters responsible for nonspecific cation transport in Trk-deficient cells have identified a number of permease transporters. These transport a diverse range of molecules and nutrients (Ko et al. 1993; Wright et al. 1997; Liang et al. 1998; Madrid et al. 1998).
Recently there has been a report linking permeases and chemotherapeutic drug uptake in S. cerevisiae. A screen to identify mechanisms of bleomycin resistance identified Sky1, Ptk2 and Agp2 as determinants of sensitivity (Aouida et al. 2004). Loss of these factors increased resistance to bleomycin. Ptk2 kinase positively regulates Pma1, and Sky1 may negatively regulate Trk1,2 (Forment et al. 2002) and so these phenotypes are consistent with predictions made from our studies on Trk1,2 reported here.
Agp2 is a transporter of L-carnitine (van Roermund et al. 1999). This study suggests that it can transport bleomycin, as uptake was reduced in an agp2 mutant and increased upon Agp2 over-expression (Aouida et al. 2004). Therefore, transporters that are normally specific for nutrients may determine uptake of chemotherapeutic agents by yeast. From our results, we would postulate that hyperpolarization of the plasma membrane would affect the binding affinity of transporters like Agp2 for different substrates, resulting in increased transport of drugs into the cells.
In this screen, disruption of neither Hal4, Hal5 nor Trk1 were identified as resulting in increased sensitivity to bleomycin. In the case of Hal4 and Hal5, only a double mutant displays a phenotype, and this screen used viable single mutants. However, in our hands, the S. cerevisiae trk1 mutant is sensitive to bleomycin (data not shown). The reason for this difference is not clear, but may be due to differences in genetic background.
The principle seen here for affecting drug sensitivity by altering the plasma membrane potential may be of therapeutic value, as potassium transport impacts on many key processes in eukaryotic cells. Altered expression of potassium channels, and a concomitant change in membrane potential, has been reported for a number of cancers and cancer cell lines (Arcangeli et al. 1995; Bianchi et al. 1998; Cayabyab & Schlichter 2002; Mu et al. 2003; Pei et al. 2003). However, little or no work has been done to determine the effect of this altered potential on the susceptibility of tumors to chemotherapeutic treatment, or whether the effectiveness of therapy could be increased using channel openers/blockers to modulate this altered potential.
In summary we show that fission yeast Hal4 acts as a regulator of membrane potential possibly through the Trk1,2 potassium transporter, and that this is important not only for salt homeostasis, but also for innate resistance to chemotherapeutic agents. However, this pathway is more complex and further work is required to tease apart the underlying mechanisms.
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Experimental procedures |
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E. coli (XL1-Blue) were grown using standard procedures, and transformations performed by electroporation. All S. pombe strains used are listed in Table 1. S. pombe was grown and manipulated using standard conditions (Moreno et al. 1991). For potassium starvation experiments mineral media was used (Calero et al. 2000). Concentrations of ammonium phosphate and ammonium sulfate were 30 mM and 8 mM, respectively, and the vitamins used in standard EMM were used at 5x concentration. S. pombe cells were transformed using a modified version of the Lithium Acetate procedure or by electroporation (Suga & Hatakeyama 2001).
Isolation of hal4-1
Wild-type S. pombe were plated to a density of 3 x 103cells/plate and exposed to a predetermined dose of UV calculated to give one lesion per cell. Sensitivity acquired due to mutation was selected by replica plating resultant colonies to yeast extract plates with and without 0.5 µg/mL bleomycin (Fluka) and selecting colonies with acquired sensitivity. Wild-type forms colonies up to approximately 5 µg/mL bleomycin.
Disruption and epitope tagging of trk1+, trk2+ and hal4+
All strains used are derived from h+ade6M-210 ura4-D18 leu1-32 his7-366. Gene disruptions and epitope tagging were carried out using the one step method (Bahler et al. 1998). For all strains, homologous recombination was confirmed by PCR analysis. All oligonucleotide sequences are available on request.
Cloning of hal4+
A culture of the hal4-1 mutant was grown to log phase. 1 x 109 cells were transformed with 1 µg pTN-L1 genomic library DNA by electroporation (Nakamura et al. 2001). Samples were diluted with an equal volume of 1 M sorbitol and 50 µL spread per minimal plate containing all amino acid supplements with the exception of leucine. These were replica-plated to plates containing calcium for selection of clones that rescued sensitivity. Complete rescue of all drug phenotypes was subsequently confirmed.
Determination of sensitivity of yeast strains
Drug sensitivity was determined using dilution assays. Briefly, 5 µL of a cell suspension containing approximately 2 x 106 cells/mL was pipetted on to yeast extract plates containing appropriate ranges for each drug. In some cases, an extra control plate containing DMSO was required for those drugs that were not water-soluble, and for the trk1trk2 mutant, 10 mM KCl was present in all plates used.
Confocal imaging of doxorubicin uptake
Log phase cells were diluted to give 2 x 106 cells/mL, using yeast extract prewarmed to 30 °C and grown for 1–1.5 generations. Doxorubicin was added to a final concentration of 40 µg/mL and cells grown for 2 h. A 1.5 mL aliquot was taken, pelleted at 2 k for 2 min, and washed twice in 1 mL water to remove excess doxorubicin. Cells were re-suspended in water and visualized using a Zeiss Laser Scanning Confocal Microscope mounted on a Zeiss AxioVert 100 M and viewed via x 63 plan-Apochromat 1.4 NA objective lens. Excitation of the fluorophore was achieved via a 20 mW Argon Ion Laser, 5% of the available laser power was utilized to excite the sample. A Rhodamine long pass filter was then placed in the light path so that light above 570 nm was recorded on the photomultiplier.
Confocal imaging of membrane potential state
This was based on a fluorimetric method previously reported for measuring membrane potential in S. cerevisiae (Gaskova et al. 1998, 1999, 2001).
Wild-type and hal4-1 mutant cells were grown to mid log phase in yeast extract, washed twice with 1 mL water and once in 10 mM citrate-phosphate (CP) buffer pH 5.6. Cells were re-suspended in 1 mL CP buffer and 15 µL of this was mixed with 15 µL soybean lectin (0.1 mg/mL) on a glass chamber slide in a Petri dish with a damp tissue and left for 10 min. Excess cells were removed by gently washing over CP buffer. The slide was placed in the chamber and assembled according to the manufacturers’ instructions. 1 mL of CP buffer was added and the confocal laser positioned in the correct plane of focus. The buffer was replaced with 1 mL CP buffer containing 0.1 µg/mL DiSC3(3). The dye was excited at 514 nm using a HeNe laser at 15% and emissions recorded every 15 s through planes of the cells over 25 min (mutant) or 30 min (wild-type).
Potassium starvation
Cells were grown to a density of 0.5 x 106 cells/mL. They were washed once with sterile water and re-suspended at the same density in mineral media. Cells were then incubated at 30 °C with agitation and samples collected every hour for 4 h.
Western analysis
Cell cultures were grown to early log phase and washed once in 1 mL stop buffer (Simanis & Nurse 1986). Crude extracts were prepared from 2.5 x 108 cells resuspended in 100 µL cell lysis buffer (50 mM Tris, 150 mM NaCl, 2% NP-40 and one mini protease inhibitor tablet (Roche) per 10 mL), and cells were lyzed using glass beads. The protein extract was adjusted to contain 1% NP-40, and 5 mg were loaded on to the matrix. Protein was immunoprecipitated using anti-HA affinity matrix (Roche), according to the manufacturers’ instructions. The matrix was washed three times with lysis buffer containing 1% NP-40, and protein eluted from the resin by boiling for 5 min in 30 µL SDS loading buffer.
For immunological detection, proteins were transferred to PVDF membranes (Immobilon P, Millipore), blocked and detected with 1 : 1000 dilution of mouse anti-HA antibody (CRUK). Immunoreactive proteins were identified using a horseradish peroxidase–linked mouse immunoglobulin and luminescence substrates (ECLplus; Amersham Biosciences).
Phosphatase treatment of Trk1 HA tagged protein
Crude extracts were obtained from exponentially growing cells and 1 mg total protein loaded on to anti-HA matrix and immunoprecipitated as before. Prior to removal from the beads, a 20 µL reaction mix containing lambda phosphatase (New England Biolabs) was added to each sample. Reactions both with and without enzyme were set up and rotated at room temperature for 30 min 20 µL SDS loading buffer was then added to each sample and these incubated at 100 °C as before to elute bound protein.
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Footnotes |
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* Correspondence: E-mail: njones{at}picr.man.ac.uk
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Received: 12 May 2005
Accepted: 26 June 2005
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