P pak4 ser474

Cells treated with or without E₂ were seeded on coverslips and cultured in DMEM/10% FBS overnight. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with goat serum before incubation with antibodies against Pak4 (1:100, Abcam), or p-Pak4 Ser⁴⁷⁴ (1:100, Cell Signaling Technology) at 4°C overnight. The cells were incubated with Alexa 594-labeled secondary antibodies (1:200; Invitrogen, Burlington, ON, Canada), and counterstained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime) before analysis using a microscope (Leica TCS SP8). The control slides were treated with PBS instead of the primary antibody.

Cells were lysed in RIPA buffer (Beyotime Biotechnology, China) containing protease and phosphatase inhibitors, and the total protein was quantified by BCA assay (Beyotime Biotechnology, China). The subsequent Western blotting analysis was carried out according to the usual procedure 22 (link). The primary antibodies used are listed as follows: p-VEGFR2-Y1175 (AP0382, Abclonal, China); Tubulin (AC008, Abclonal, China); p-JNK1/2/3 (AP0631, Abclonal, China); JNK1/2/3 (A11119, Abclonal, China); p-PAK2(Ser20) (AP0803, Abclonal, China); p-PAK4(Ser474) (3241S, Cell Signaling Technology, USA); RhoJ (H00057381-M01, Abnova, Taiwan, China); moesin (M7060, Sigma, USA); GAPDH (A19056, Abclonal, China); β-actin (AC026, Abclonal, China); Braf (14814, Cell Signaling Technology, USA); p-Braf (Ser445) (2696, Cell Signaling Technology, USA); ERK1/2 (9102, Cell Signaling Technology, USA); p-ERK1/2 (4370, Cell Signaling Technology, USA).