Western Blot for IL-13Rα2 Protein Detection

Harvested cells were lysed with standard RIPA lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% NP-40, 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate) containing 1% mammalian proteinase inhibitor mix. Cell lysates fractionated by SDS-PAGE gel electrophoresis were transferred onto polyvinylidene fluoride (PVDF) membranes as described [39 (link)]. IL-13Rα2 protein was detected with an anti-IL-13Rα2 mouse monoclonal antibody (sc-134363, Santa Cruz Biotechnology) diluted 1:500 in blocking buffer (TBS, 0.1% Tween-20 containing 5% milk powder) followed by incubation with an HRP-conjugated anti-mouse secondary antibody. Where indicated, mCherry was detected with rabbit anti-RFP antibody (Abcam [#ab34771]). Membranes were developed using ECL Select Substrate (GE Healthcare (#RPN2235), Hatfield, UK) and bands were visualized using a Bio-Rad ChemiDoc TM XRS system (Watford, UK). For standardization, blots were stripped as described [40 (link)] and re-probed using an anti-α tubulin mouse monoclonal antibody (Merck (#T6074), Gillingham, UK).