Anti flag rabbit monoclonal antibody

Cells grown on coverslips were fixed with Immunol Staining Fix Solution at room temperature for 5 minutes and blocked in QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime P0260) at 37 °C for 30 minutes. Then, the cells were washed with phosphate-buffered saline (PBS, pH 7.2) for 30 min and incubated with primary antibodies at a 1:100 dilution in 5% bovine serum albumin (BSA) at 4 °C overnight, followed by incubation with secondary antibodies at a 1:200 dilution at 37 °C for 1 h and Hoechst 33342 staining at room temperature for 5 minutes. Finally, the cells were mounted with Antifade Mounting Medium (Beyotime Biotechnology, P0128) and examined under a fluorescence microscope (Olympus, IX73, Japan). The primary antibodies used in this study were mouse anti-Stat3 monoclonal antibody (Cell Signaling Technology, 9139, USA), rabbit anti-Flag monoclonal antibody (Cell Signaling Technology, 14,793), and rabbit anti-c-Myc monoclonal antibody (Cell Signaling Technology, 18,583) and the secondary antibodies were donkey anti-rabbit IgG (H + L) Alexa Fluor Plus 488 (Invitrogen, A32766, USA) and donkey anti-mouse IgG Alexa Fluor Plus 555 (Invitrogen, A32773).

Membrane lipid binding analysis of Flag-tagged FERM or mutant was conducted using PIP-StripsTM membranes (Thermo Fisher Scientific), with each nitrocellulose membrane containing 100 pmol of 15 different phospholipids and a blank sample. PIP strip was blocked in TBST with 3% fatty acid–free BSA for 1 hour at RT, followed by adding Flag-tagged FERM (5 μg/ml) or mutant at 4°C overnight. The membrane was then washed three times with TBST and blotted with an anti-Flag rabbit monoclonal antibody (1:1000; Cell Signaling Technology, catalog no. 14793S) overnight at 4°C. After washing again three times with TBST, the membrane was then incubated with the appropriate species-specific HRP-conjugated anti-IgG secondary antibody (1:5000; Beyotime Biotechnology, catalog no. A0208) for 1 hour at RT. Proteins were detected using a chemiluminescence imaging system (Clinx Science).