FIGURE 5.

Distribution of tyrosine 23 and serine 25 double phosphorylation mutants of AnxA2 in response to glutamate treatment. A–D, 661W cells were transiently transfected with plasmid vectors expressing phosphomimetic and non-phosphomimetic double mutants at tyrosine 23 and serine 25 (AnxA2Y23FS25A-GFP, AnxA2Y23FS25E-GFP, AnxA2Y23ES25A-GFP, and AnxA2Y23ES25E-GFP). The cells were treated with 500 μm glutamate for 4 h. The EDTA eluates and cell surface biotinylated extracts were collected as mentioned previously and immunoblotted with anti-GFP antibody. The EDTA eluates were tested for their purity by immunoblotting with anti-PGK antibody. A Coomassie-stained gel was used as a loading control for the EDTA eluates. The biotinylated extracts were probed with anti-Na,K-ATPase antibody to determine equal loading. WCL, whole cell lysate; Biot., biotinylated extracts. All the blots were exposed to identical exposure times.