Fig. (2).

The effect of phosphorylation of TRPV4 serine 824 on the interaction between TRPV4 and STIM1. (A) Co-immunoprecipitation of TRPV4 WT, S824A, or S824D with STIM1. HEK 293 cells were transiently transfected with EGFP-TRPV4 WT or S824A plasmid. After 48 h the cells were lysated, and pull-down of total protein was conducted with protein Aagarose beads. Western blot assays were performed with rabbit anti-TRPV4 or anti-STIM1 antibody. (B) Pull-down analysis of STIM1 with GST fusion C-terminal TRPV4 WT, S824A, or S824D. GST-fusion proteins encompassing C-terminal TRPV4 domains were constructed and expressed in E. coli. Approximately 2 mg of WT, S824A or S824D fusion protein bound to the glutathione-Sepharose bead were incubated with HEK 293 cell lysates.