Cells were collected and lysed in M2 buffer (20 mM Tris, pH 7, 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulphonyl fluoride, 20 mM glycerol phosphate, 1 mM sodium vanadate and 1 μg ml−1 leupeptin). Cell lysates were separated by SDS–PAGE and analysed by immunoblotting. For non-reducing gel analysis, cells were lysed in M2 buffer without DTT and separated by SDS–PAGE without β-mercaptoethanol. The dilution ratio of the antibodies used for western blotting is 1:1,000. The proteins were visualized by enhanced chemiluminescence, according to the manufacturer’s (Amersham) instructions.
For immunoprecipitation, lysates were precipitated with antibody (1 μg) and protein G-agarose beads by incubation at 4 °C overnight. Beads were washed four to six times with 1 ml M2 buffer, and the bound proteins were removed by boiling in SDS buffer and resolved in 4–20% SDS–polyacrylamide gels for western blot analysis.
For TRPM7 endogenous immunoprecipitation, crosslinking of cellular proteins was performed before the cell lysis. Cells were washed three times with ice-cold PBS solution and incubated with a crosslinking reagent (DSP, 2 mM) in PBS for 30 min at room temperature, followed by incubation in 10 mM Tris, pH 7.5, buffer for 15 min to quench the reaction. Then, cells were lysed in RIPA buffer containing 0.1% SDS and the samples were used in regular immunoprecipitation procedure as described above. All western data are representative of two or three independent experiments.
For immunoprecipitation, lysates were precipitated with antibody (1 μg) and protein G-agarose beads by incubation at 4 °C overnight. Beads were washed four to six times with 1 ml M2 buffer, and the bound proteins were removed by boiling in SDS buffer and resolved in 4–20% SDS–polyacrylamide gels for western blot analysis.
For TRPM7 endogenous immunoprecipitation, crosslinking of cellular proteins was performed before the cell lysis. Cells were washed three times with ice-cold PBS solution and incubated with a crosslinking reagent (DSP, 2 mM) in PBS for 30 min at room temperature, followed by incubation in 10 mM Tris, pH 7.5, buffer for 15 min to quench the reaction. Then, cells were lysed in RIPA buffer containing 0.1% SDS and the samples were used in regular immunoprecipitation procedure as described above. All western data are representative of two or three independent experiments.
