Mouse anti gapdh 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 cell lysis buffer (Beyotime, China) was used to harvest the EPCs proteins. Then, SDS-PAGE was run to isolate the extracted proteins, followed by transferring the proteins onto the PVDF membranes (Millipore, USA). Typically, the rabbit anti-phospho-ERK, rabbit anti-CXCR7, and rabbit anti-ERK antibodies (all 1:1000; Abcam, USA), and the mouse anti-GAPDH antibody (1:5000; Abbkine, China) were used. Later, the HRP-labeled anti-rabbit IgG and anti-mouse IgG (both 1:5000; Beyotime, China) were used for visualizing the proteins, and ECL chemiluminescence system (Tanon, China) was used for color development.