Anti rabbit hrp secondary antibodies

Western blotting was performed as previously described [21 (link)]. Cells’ total protein was extracted with lysis buffer (Invitrogen), and the target proteins were separated from total protein by SDS/PAGE. The target proteins on gel were transferred to the PVDF membrane (Millipore). BSA (Gibco) sealed up PVDF membrane for 2 h, proteins were marked by primary antibodies anti-p65 antibody (1:1000, Abcam) or anti-p50 antibody (1:10000, Abcam) or anti-β-actin antibody (1:2000, Abcam) and anti-rabbit HRP secondary antibodies (1:1000, Abcam). PVDF membrane was washed with PBS and we observed the chemiluminescence with ECL Plus Western Blotting Substrate (Thermo Fisher). The film that was placed on the PVDF membrane was exposed and developed.

Western blotting assays were conducted according to our previous description [20 (link)]. Briefly, cells were collected and lysed in RIPA buffer (Invitrogen, USA), and total proteins were isolated. A BCA assay kit was utilized in order to determine the concentration of the protein, and SDS-PAGE was utilized in order to distinguish the target proteins from the overall protein. Following that, the proteins were deposited onto PVDF membranes (Millipore). Bovine serum albumin (Gibco, USA) was used to block the PVDF membrane for 2 h at room temperature, followed by incubation with primary antibodies against STAT3 (1 : 1000, Abcam) or GAPDH (1 : 2000, Abcam) at 4°C overnight and then with antirabbit HRP secondary antibodies (1 : 1000, Abcam) at 37°C for 1 h. Detection was performed with enhanced chemiluminescence (ECL) reagents. ImageJ software was utilized in order to do statistical analysis and quantify the obtained data. For the purpose of determining relative protein expression, the ratio of densitometry readings to the values that correspond to GAPDH was utilized.