Anti p akt antibody

Proteins were extracted from liver tissues and LO2 cells using RIPA lysis buffer supplemented with PMSF (Beyotime Institute of Biotechnology, China). Protein samples were electrophoresed, then transferred onto PVDF membranes (MilliporeSigma, USA), and blocked with 5% nonfat milk at room temperature for 1 h. Then the PVDF membrane was incubated with the primary antibody solution anti- RBM15 antibody (1: 1000, Proteintech, China),anti-PI3K antibody (1: 1000; CST, USA),anti-ERK antibody (1:1000, CST, USA), anti-p-ERK antibody (1:1000, Wanlei, China), anti-AKT antibody (1:1000; Proteintech, China),anti-p-AKT antibody (1:1000; Proteintech; China) and anti-β-ACTIN (1:1000; Servicebio, China) overnight at 4 °C. Monoclonal anti-rabbit IgG (1:5000, ZSGB-Bio, China) or monoclonal anti-mouse IgG (1:5000, ZSGB-Bio, China) were incubated with the PVDF membrane for 1 h at room temperature. Finally, enhanced chemiluminescent substrate (MilliporeSigma, USA) was used to detect blots by a ChemiDoc XRS + system (Bio-Rad, USA).

RA-FLSs were lysed using ice-cold lysis buffer, and total proteins were extracted and quantified using a protein quantification assay. Equal volumes of protein were mixed with loading buffer and resolved by performing SDS-PAGE. Resolved proteins were transferred to polyvinylidene fluoride membranes and then incubated with the following primary antibodies: anti-PTEN antibody (Abcam, 1:1000), anti-AKT antibody (Cell Signaling, 1:2000), and anti-p-AKT antibody (Proteintech, 1:2000). The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies and the binding signal was visualized using an enhanced chemiluminescence system (GE Systems).

The protein expressions were detected by western blotting. Cell samples were homogenized with cell lysis buffer (Cell Signaling Technology, USA) and the supernatant was collected centrifugally. The samples were separated on 10% or 8% polyacrylamide-SDS gel and electrically imprinted on polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Then, the PVDF membranes were blocked in 5% non-fat milk in tris-buffered saline with Tween 20 (TBST) for 1 h and incubated with primary antibodies at 4 °C overnight. After washing, the PVDF membranes were incubated with the secondary HRP conjugated secondary antibodies (Proteintech, USA) at 1:5000 for 2 h at room temperature. Protein blots were detected by an ECL western blotting kit (Proteintech, USA) and quantitated by ImageLab software. The details of the primary antibodies in this experiment were presented as follows: anti-β-actin antibody (Proteintech, USA), anti-p-AKT antibody (Proteintech, USA), anti-AKT antibody (Proteintech, USA), anti-PIK3CA antibody (Abclonal, China), anti-p-PI3K antibody (Abclonal, China), anti-VEGF antibody (Proteintech, USA), and anti-TET2 antibody (Proteintech, USA).