Immunoblotting analysis was conducted as previously described 33 (link). Compound-treated cells were resuspended in RIPA lysis buffer. After centrifugation at 16,200 g for 10 min at 4 °C, supernatants were collected in a pre-cooled microfuge tube. Protein concentration was determined by the BCA Protein Assay. Equal amounts of protein were loaded into 10% SDS-PAGE, and proteins were further transferred onto PVDF membranes after electrophoresis. Membranes were blocked with 5% FBS in TBST for 1 h at room temperature followed by incubation with primary antibodies, including antibodies against CDK4, CDK6, β-actin (Santa Cruz Biotechnology, sc-23896, sc-7961, sc-8432), BCL-2 (BD Biosciences, 560637), cyclin D1, p-Rb, Rb, Ac-Histone H3, caspase-3, p-JAK1, JAK1, p-STAT3 and STAT3 (Cell Signaling Technology, CST# 2978, 8516, 9309, 8173, 9662, 3331, 3332, 9145, 4904) overnight at 4 °C. Membranes were washed with TBST three times followed by incubation in HRP-labelled secondary antibodies for 1 h at room temperature. Signal was detected using Millipore Immobilon ECL reagent.
Protein signal density was quantified using ImageJ software (Version 1.60, National Institutes of Health, USA) and subsequently labelled under the bands. All lanes were normalized by dividing the density by the density of β-actin.
Protein signal density was quantified using ImageJ software (Version 1.60, National Institutes of Health, USA) and subsequently labelled under the bands. All lanes were normalized by dividing the density by the density of β-actin.
