Fig. 8
RA-GEF was basally phosphorylated by WNK1. a (Left) Total halo-WNK1 was detected with anti-halo. Actin was a loading control. (Middle) Control or WNK1-expressing BAF cells with or without OA were analyzed by Phos-tag (upper) or conventional (lower) SDS-PAGE followed by immunoblotting with anti-RA-GEF-2. (Right) Quantification of ①, ②, and ③ bands, which is presented as percentage of each band. *1P < 0.01 versus control cells with OA. b (Left) Total halo-WNK1 was detected with anti-halo. Actin was a loading control. (Middle) Flag-RA-GEF-1, and control or WNK1-expressing 293T cells with or without OA were analyzed by Phos-tag (upper) or conventional (lower) SDS-PAGE followed by immunoblotting with anti-flag. (Right) Quantification of ①, ②, and ③ bands, which is presented as percentage of each band. *1P < 0.002, *2P < 0.002 versus control cells in the presence or absence of OA. c (Left) Total WNK1 was detected with anti-WNK1. Actin was a loading control. (Middle top) Control or WNK1-knockdown (KD) BAF cells with or without OA were analyzed by Phos-tag (upper) or conventional (lower) SDS-PAGE followed by immunoblotting with anti-RA-GEF-2. (Bottom) Quantification of the abundance of ①, ②, and ③ bands, which is presented as percentage of each band. *P < 0.05 versus control cells in the presence of OA. (Right top) Control or WNK1KD, flag-RA-GEF-1-expressing BAF cells with or without OA were analyzed by Phos-tag (upper) or conventional (lower) SDS-PAGE followed by immunoblotting with anti-flag. The Phos-tag blot of flag-RA-GEF-1 was cropped from a same blot. (Bottom) Quantification of ①, ②, and ③ bands, which is presented as percentage of each band. *1P < 0.009, *2P < 0.008 versus control cells in the absence or presence of OA. The Rf value of 1.0 is defined as the position of the BPB dye. A representative of three independent experiments is shown. Each bar graph represents the means ± SEM