Mouse anti flag antibody

Hela cells in 6-well plates were washed three times with PBS and then lysed by radio-immune precipitation assay (RIPA) with lysis buffer(1% Triton X-100, 1% deoxycholate, 0.1% SDS) (Beyotime, China).The protein concentration was quantified by BCA Protein Assay Kit (Beyotime,China) and the total proteins were subjected to SDS-PAGE and then transferred to a polyvinylidenefluoride (PVDF) membrane (Millipore, USA). After blocking with 5% skim milk or 5% Bovine serum Albumin (BSA) in TTBS buffer (1L TTBS contains 8.77g NaCl, 2.42g Tris, 1g Tween, pH7.5) for 2 hours, the membrane was incubated with primary antibodies overnight at 4°C before it was washed with 1xTTBS for 3 times, then it was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour followed by wash with 1xTTBS for 3 times, 10 minutes each. Protein bands were detected by enhanced chemiluminescence (ECL) kit (Millipore, USA) and visualized by LAS4000 mini (GE, USA). The primary antibodies used in this study including: mouse anti-flag antibody, mouse anti-GAPDH antibody and mouse anti-β-Actin (Abbkine, Redlands, CA), rabbit anti-STAT1, rabbit anti-Phospho-STAT1 (Y701) (Cell Signaling Technology, Inc., Beverly, MA). The secondary antibodies were HRP-conjugated ECL goat anti-rabbit IgG, or HRP-conjugated ECL sheep anti-mouse IgG (Abmart, China).

The transfected cells were fixed in 4% paraformaldehyde. After washing three times, they were incubated in a blocking solution (10% goat serum/PBS/0.25% Triton X-100) for 1 h. The cells were then incubated with 1:400 mouse anti-FLAG antibody (# A02010, Abbkine) in the blocking solution at 4°C overnight. After washing, the cells were incubated with 1:500 Alexa Fluor 488-conjugated goat anti-mouse IgG (A11034, Molecular Probes) in PBS at room temperature for 1 h. After washing three times and staining with DAPI, the slides were observed with a confocal microscope.