Ab64148

After 48 hours of transfection, the cells in six-well plates were collected and placed on ice. To extract the proteins, RIPA lysate with protease inhibitor was used. BCA method was used to determine the protein concentration. Then we added about 20 μg protein to each well of a vertical electrophoresis tank after being heated at 95 °C for 5 min. Following that, the protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. The primary antibodies used in the current study were list as follows: ARHGEF39 (1:1000, cat.no. ab67211, Abcam), AKT (1:500, cat. no. ab64148, Abcam), p-AKT (1:500, cat.no. ab8932, Abcam), ERK (1:1000, cat.no. ab32537, Abcam), p-ERK (1:1000, cat.no. ab131438, Abcam). Following that, the membrane was rinsed with TBST 35 min and incubated with suitable secondary antibodies at ambient temperature. Then the protein bands were washed and developed with enhanced chemiluminescence western blot detection kit. The gray value was scanned by the QUANTITY ONE software and the relative expression of each protein was calculated with GAPDH as the internal reference.

Proteins were obtained using whole cell lysis buffer (Roche, Basel, Switzerland) for 30 min on the ice. Protein samples were quantified using Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Then, 30 µg of proteins were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% w/v nonfat dry milk for 1 h, primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody (ab205718; Abcam, Cambridge, MA, USA). The protein bands were measured using the enhanced chemiluminescent (ECL) system (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Gray analysis was conducted to quantify the expression of proteins using ImageJ software. Primary antibodies, including anti-BRD4 (ab128874), anti-phosphorylated-phosphatidylinositol 3-kinase (anti-p-PI3K; ab70912), anti-PI3K (ab32089), anti-p-AKT serine/threonine kinase (p-AKT; ab38449), anti-AKT (ab64148), anti-CD63 (ab59479), anti-CD81 (ab79559) and anti-β-actin (ab8226), were purchased from Abcam.

Total extracted proteins were separated by electrophoresis and were subsequently transferred onto polyvinylidene fluoride membranes. After electrophoresis, the membranes were incubated with the below primary antibodies: anti-GAPDH (#ab9483; Abcam), anti-p38 (#ab27986; Abcam), anti-phosphorylated (p)-p38 (#ab236527; Abcam), anti-STAT3 (#ab68153; Abcam), anti-p-STAT3 (#b32143; Abcam), anti-AKT (#ab64148; Abcam), and anti-p-AKT (cat. no. ab8933; Abcam). Then, the membranes were incubated with relative secondary antibodies at room temperature for 2 h.