Protein Extraction and Western Blotting

Proteins were extracted using RIPA Lysis buffer containing Phosphatase Inhibitor Cocktail (Sigma Aldrich, Sydney, Australia) according to the manufacturer’s instructions. Protein concentration was quantified using a BCA Protein Determination Kit (Pierce) as per the manufacturer’s instructions and absorbance read at 562 nm on a PerkinElmer VICTOR X Multilabel Plate Reader. Western blots were performed as previously described [19 (link)], utilizing rabbit antibodies against phospho-STAT3 (Tyr 705), total-STAT3, BCL2 and ICAM-1 (Cell Signaling, Beverly, MA, USA), and a mouse monoclonal antibody against GAPDH (Millipore Technologies, Sydney, Australia) at 1:1000 dilution, followed by anti-rabbit IRDye™ 680 CW and anti-mouse IRDye™ 800 CW (LI-COR Biosciences, Nebraska, USA) at 1:10000 dilution in 5% (w/v) BSA in TBS containing 0.1% Tween. Images were obtained using an Odyssey Imaging System (LI-COR Biosciences) and analysed with ImageJ (NIH).