Western Blotting for Protein Quantification

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.