Western blotting luminol reagent

Western blot was performed as described previously [16 (link)]. Briefly, cells were lysed on ice for 5 min in 2 × SDS buffer with β-mercaptoethanol, and were treated in 95 °C for 10 min. Supernatants were harvested by centrifugation for 1 min at 10,000×g. Total proteins were loaded and separated on SDS-PAGE gels, and were electroblotted onto PVDF membrane. The membrane was soaked for 2 h in block solution, and then incubated overnight in the presence of rabbit polyclonal to signal transducers and activators of transcription 3 (STAT3, phospho Y705, ab76315), or STAT3 (ab32500) (Abcam, Cambridge, MA, USA; 1: 1000 dilution). Horseradish peroxidase-conjugated goat anti-rabbit antibody IgG (Abcam; 1: 2000 dilution) was added for additional 2 h incubation. Antigen-antibody complexes were observed by enhanced chemiluminescence (Western Blotting Luminol Reagent, Cell Signaling Technology, Danvers, MA, USA).

The cells were lysed in RIPA Lysis Buffer (20-188, Millipore, Burlington, VT, USA) containing phosphatase inhibitor cocktail tablets (04906837001, Roche, Basel, Switzerland) and protease inhibitor cocktail tablets (11836153001, Roche, Basel, Switzerland). The protein concentration was measured with Protein Assay Dye Reagent Concentrate (cat. no. #5000006, BIO-RAD, Hercules, CA, USA). For a Western blot analysis, equal amounts of protein were separated on 8–15% sodium dodecyl sulfate (SDS)-polyacrylmide gels, and the separated protein was transferred to Amersham™ Protran™ 0.2 µm NC (10600001, Amersham™; Cytiva, Pittsburgh, PA, USA). After blocking the transferred membrane at room temperature by using phosphate-buffered saline (PBS) buffer containing non-fat milk (10%) and Tween 20 (0.1%) for 1 h, the membranes were treated with specific antibodies overnight in a cold chamber. After washing with Tris-buffered saline (cat. no. A0027, BIO BASIC, Markham, ON, Canada), the membranes were treated with an HRP-linked secondary antibody (Anti-rabbit IgG cat. no. 7074S or Anti-mouse IgG cat. no. 7076S; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h at room temperature, and visualized by Western Blotting Luminol Reagent (cat. no. sc-2048, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).