1 × 107 of HEK-293 cells expressing TRPM6 and TRPM7 were plated for in vitro phosphorylation experiment. To analyze protein expression levels of DT40 mutant cells, 0.1 to 0.5 × 107 DT40 mutant cells were plated and expression of the indicated molecules was induced by adding doxycycline to the growth media for 24–48 hours. HEK-293 or DT40 cells were lysed using standard protocols. Anti-Flag (Sigma) and anti-HA (Roche) immunoprecipitations were performed with cell lysates. Bound proteins from whole cell lysates or immunoprecipitated proteins were washed 3 times with lysis buffer, then separated by SDS/PAGE using 6% polyacrylamide gels, and transferred to a PVDF membrane. The membranes were analyzed by Anti-Flag (Sigma), anti-HA (Roche) or a general anti-P-Ser antibody (from Cell Signaling, cat.# 2981) immunoblotting, as indicated. Blots were sometimes re-probed after stripping.
Anti p ser antibody
Manufactured by Cell Signaling Technology
The Anti-P-Ser antibody is a laboratory tool used to detect and analyze phosphorylated serine residues in proteins. It provides a reliable method for identifying and studying post-translational modifications involved in cell signaling pathways.
Automatically generated - may contain errors
2 protocols using anti p ser antibody
1
Quantitative Analysis of TRPM6/7 Phosphorylation
2
Antibody-based detection of TRPM7 and TRPM6
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For detection of proteins, the proteins resolved by SDS-PAGE and western blotting using standard protocols. A rat monoclonal antibody anti-HA antibody (3F10; Roche Life Sciences, IN) was used to detect HA-tagged TRPM7 and TRPM6. Anti-FLAG M2 antibody (Sigma, MO) was used to detect FLAG-TRPM7. A mouse monoclonal antibody anti-SBP (sc-101595; Santa Cruz Biotechnology, Inc., TX) and a rabbit polyclonal anti-TRPM7-C47 were used to detect SBP-TRPM77 (link). An anti-GFP antibody (sc-9996; Santa Cruz Biotechnology, Inc., TX) was used to detect GFP-TRPM7-Cterm. An anti-pSer antibody (#2981; Cell Signalling Technology, Inc., MA) was used to detected phosphorylated TRPM6 protein.
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