Hrp labeled goat anti rabbit secondary antibody

The protein levels of Leptin-R, VEGFR-1, STAT3, p-STAT3, and BCL-2 after drug treatments were evaluated using Western immunoblotting. The total protein was extracted from the tumor tissues, and the total protein concentration was detected by the BCA Protein Concentration Assay Kit. Proteins were separated using SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) for 2 h and then washed with TBST three times, each for 5 min on a shaker. The primary antibodies used were Leptin-R (Abcam 1:2000), STAT3 (CST 1:2,000), p-STAT3 (CST 1:1,000), BCL-2 (Abcam 1:2,000), VEGFR-1 (Abcam 1:2,000), and β-actin (Abcam 1:2,000). After incubation overnight at 4°C with primary antibodies, the membranes were washed with TBST and then incubated with one of the following secondary antibodies at room temperature for 1 h: HRP-labeled goat anti-rabbit secondary antibody (Jackson 1: 2,000) or HRP-labeled goat anti-mouse secondary antibody (Jackson 1:2,000). The immunoblots were then visualized using enhanced chemiluminescence (ECL) reagents and exposed to X-ray film. The membranes were subsequently re-probed with anti-β-actin antibody. Western blots were performed according to the manufacturer’s instructions with standards run in triplicate. The gray values of the immunoblots were semi-quantitatively determined by image analysis.