Cocktail set 3

Cells were lysed with ice-cold RIPA lysis buffer (Bio Basic, Markham, ON, Canada) containing freshly added phosphatase (halt phosphatase inhibitor; Thermo Scientific, Waltham, MA, USA) and protease inhibitors (cocktail set III, Calbiochem, Merck Millipore, Burlington, MA, USA). Western blotting was carried out using standard protocols. Antibodies against α-SMA and p21^Waf1/Cip1 were purchased from Dako (Santa Clara, CA, USA; M0851) and Merck (Boston, MA, USA; clone CP74, 05-655) respectively, whilst antibodies against LC3B (ab51520), p62 (ab56416), SIRT1 (ab32441) and GAPDH (ab9485) were obtained from Abcam (Cambridge, UK). These antibodies have been widely used and characterised and were shown to be specific in Western blots (Supplementary Fig. S7). Bound antibodies were detected with WesternBright Sirius enhanced chemiluminescence (ECL) reagent (Advansta, San Jose, CA, USA) using an Odyssey FC Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Densitometric analyses were conducted using ImageJ software^{55 (link)}.

Cells were lysed in ice-cold NP40 lysis buffer (150mM NaCl, 1% IGEPAL® CA-630, 50mM Tris-HCl (pH 8.0)) containing protease inhibitors (cocktail set III; Calbiochem, Merck Millipore, Darmstadt, Germany) and phosphatase inhibitors (Halt phosphatase inhibitor cocktail; Thermo Scientific, Waltham, USA). The primary antibodies used in this study were anti-DDR1 (DIG6 XP; 1:1000; Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH (ab371681; 1:1000; Abcam, Cambridge, UK). Bound antibodies were detected with peroxidase conjugated secondary antibodies and enhanced chemiluminescence reagents (Advansta, Menlo Park, CA, USA). Signal intensities were measured using ImageJ software.

HLECs were lysed in ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1% IgePal-630, 0.5% deoxycholate, 1 mM EDTA) containing a protease inhibitor mixture (Cocktail Set III) and phosphatase inhibitors (Cocktail Set V) (EMD Millipore, Billerica, MA). Following centrifugation, whole cell supernatants were collected and stored at -80˚C. Protein concentration was measured using the BCA assay (Pierce). Equal protein amounts (4–6 μg) from the stimulated HLEC lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE 4–12% Bis-Tris or 3–8% Tris-Acetate mini-gels (Invitrogen) under denaturing conditions. The separated proteins were electroblotted onto polyvinylidene difluoride membranes (pore size, 0.45 μm; Invitrogen). Protein blots were blocked for 1 h in TBS containing 0.1% Tween 20 (TBST) with 5% nonfat dry milk or 5% BSA and probed with the specific primary antibody followed by HRP-conjugated secondary antibody. Immunodetection was revealed on HyperECL film with the ECL Plus chemiluminescence detection system (GE Healthcare UK Ltd., Buckinghamshire, United Kingdom) according to the manufacturer's instructions. The relative amount of protein was estimated by densitometry analysis with Image J software (National Institutes of Health, Bethesda, MD).