Protein Expression Analysis by Western Blotting

Western blotting analysis was performed as described previously [15 (link)]. Briefly, cells treated with CT for 48 h were lysed in RIPA buffer (Beyotime, Beijing, China). The protein concentrations were quantitated with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Frederick, MD, USA). Total protein 30 µg/lane were loaded onto SDS–polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ). The membranes were blocked and incubated with (1:1000) rabbit anti-p-STAT3 (Cell Signaling, Cat: 9145L, Tyr 705), anti-STAT3 (Cell Signaling, Cat: 4904S), anti-p-JAK2 (Cell Signaling, Cat: 8082), anti-JAK2 (Cell Signaling, Cat: 3230), anti-I-kBα (Cell Signaling, Cat: 9242), anti-GAPDH (Cell Signaling, Cat: 2118) overnight at 4 °C and (1:2000) horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Cat: 70,741) for 1 h at room temperature. The protein bands were visualized using the G-BOX Chemi system (Syngene, Frederick, MD).