Lysis buffer 9803

Western blot analysis was performed as described previously (Petherick et al, 2013) using the following antibodies: GPRC5A (1:2,000, CST, 12968), β‐actin (1:10,000, Sigma, A5316), HIF‐1α (1:1,000, BD, 610959), HIF‐1β (1:1,000, BD, 611078), HIF‐2α (1:1,000, CST, 7096), PLOD2 (1:1,000, R&D, MAB4445), CA9 (1:5,000, Novus, NB100‐417), cleaved PARP (1:20,000, Abcam, ab32064), active caspase‐3 (1:1,000, CST, 96645), p‐YAP S397 (1:5,000, CST, 13619), YAP (1:5,000, CST, 14074), BCL‐XL (1:1,000, BD, 556361), BCL‐2 (1:200, Santa Cruz, SC‐509), V5‐tag (1:2,000, CST, 13202), CYR61 (1:2,000, Santa Cruz, SC‐374129), RhoA (1:2,000, CST, 2117), lamin A/C (1:10,000, Sigma, 4C11) and α‐tubulin (1:10,000, Sigma, T6199). Cells were washed with ice‐cold PBS and lysed on ice for 10 min with Cell Signaling Technology lysis buffer (9803) supplemented with protease inhibitors and sonicated briefly. Equal protein concentrations were resolved using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to an Immobilon‐P polyvinylidene difluoride membrane (Millipore). For GPRC5A Western blots, samples were not boiled.

NIH3T3 cell lysates were prepared in lysis buffer 9803 (Cell Signaling Technology) containing a cocktail of protease inhibitors (Roche, Basel, Switzerland). Keratinocyte cell lysates were prepared in a lysis buffer containing 50 mM Tris-HCl pH 7.6, 2.5% SDS, 10% glycerol, 10 mM β-glycerophosphate, 10 mM NaF, 95 µM sodium orthovanadate and a cocktail of protease inhibitors (Roche). Total protein content was determined using a BCA Protein Assay Kit (Pierce). Lysate corresponding to 10 µg total protein was subjected to PAGE using 3-8% gradient gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes using the iBlot transfer system (Invitrogen) at 12 V for 25 min. Human β1 integrin was detected using anti-human β1 integrin rabbit polyclonal antibody (Millipore, AB1952) at a 1:1000 dilution or mouse anti-human β1 integrin monoclonal antibody (BD transduction laboratories, 610467) at a final concentration of 0.1 µg/ml. Human α2 integrin was detected using anti-human α2 integrin mouse monoclonal antibody (BD transduction laboratories, BD611016) at a 1:1000 dilution. Actin was detected using a monoclonal anti-actin antibody, (Sigma-Aldrich, A4700) at 1:2000. Secondary antibodies coupled to HRP (DAKO) and ECL Western Blotting Detection Reagents (GE Healthcare) were used for visualization.

Brain homogenates prepared by Cell Signaling lysis buffer (#9803) were diluted to 5 μg/μl with PBS and plasma samples were prepared at a 2-fold dilution with PBS. Diluted samples were sent to Eve Technologies (Canada) for cytokines and chemokines measurement by using the Mouse Cytokine Array / Chemokine Array 44-Plex (MD44).