Ripa lysis buffer

The total protein was obtained by cells lysis with RIPA Lysis Buffer (Haigene, Harbin, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose (NC) membrane. Then, the membrane was placed in 5% milk for 2 h at room temperature, washed in phosphate buffer saline (PBS). The B-cell lymphoma-2 (Bcl-2, ab32124), Bcl-2 associated X protein (Bax, ab182733), cleaved caspase-3 (ab2302), β-actin (ab179467) and THBS2 (ab84469) antibodies from Abcam were diluted 1,000 times and added into membranes to incubate at 4°C overnight. And then, anti-rabbit IgG antibody (1:2,000; Abcam) was added to incubate for 30 min at room temperature. Finally, the proteins on the membrane were detected by an imaging system (Bio-Rad, Hercules, USA).

The total protein content in each sample was extracted by using a RIPA lysis buffer (Haigene, Harbin, China) containing 10 mM Tris/HCl, pH 7.4, 0.5% Triton X-100, 150 mM NaCl, and protease inhibitors according to the manufacturer’s instructions. In the next step, the extracted protein in each sample was separated on a 10% SDS-PAGE gel and blotted onto a nitrocellulose membrane, which was then blocked at room temperature for 2 h with 5% skim milk and probed with primary and secondary anti-STAT3 antibodies in sequence (Abcam, Cambridge, MA, USA) according to the recommended antibody incubation conditions provided by the manufacturer. After the membrane was washed with a TBS buffer (Sigma Aldrich, St. Louis, MO, USA) and developed by using an ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the density of the STAT3 protein bands on the membrane were assayed by using a Bio-Rad imaging system (Bio-Rad Laboratories, Hercules, CA, USA) to calculate the relative expression of STAT3 protein in each sample.