Anti calpain 1

Samples were lysed in a lysis buffer containing the following: 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulphonyl fluoride (PMSF), with protease inhibitor cocktail (pepstatin 1 g/mL, aprotinin 1 g/mL, leupeptin 1 g/mL). Protein concentrations were measured using the BCA method and 40 μg of proteins were loaded and separated using SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and blocked in 5% nonfat milk or 5% bovine serum albumin. Membranes were incubated at 4 ? overnight with the following antibodies: anti-SOD1 (1:2000, Abcam), anti-LC3 (1:1000, Sigma), anti-p62 (1:1000, CST), anti-Beclin1 (1:1000, CST), anti-mTOR (1:1000, Abcam), anti-ATG5 (1:500, MBL), anti-p62 (1:500, MBL), anti-calpain 1 (1:1000, Abcam), anti-Bip (1:1000, CST), IRE1α (1:500, CST), CHOP (1:1000, CST) and PDI (1:1000, CST), anti-cleaved-caspase-12 (1:500, CST) and anti-β-actin (1:10000, Sigma). The membranes were then incubated with appropriate peroxidase-conjugated secondary antibodies for 2 hours. Protein bands were visualized using ECL (Pierce, USA) and an image analyzer was used to quantify the densities of interested protein bands (Bio-Rad, Image lab 4.1).

Total protein extraction from human dermal fibroblasts or A549 cells was performed using an extraction kit (101 Bio, Mountain View, CA, USA). Protein concentration was determined using a spectrophotometer and bicinchoninic acid protein assay kit (TaKaRa Bio, Shiga, Japan). Aliquots of protein from each sample were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a Mini-PROTEAN TGX Precast gel and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was incubated with the appropriate primary antibody and fluorescently labeled secondary antibodies. The following antibodies were used: anti-COL1A2 (1:1000, Abcam), anti-FN1 (1:1000, Abcam), anti-αSMA (1:1000, Abcam), anti-p-Smad3 (1:1000, Invitrogen), anti-calpain 1 (1:1000, Abcam), anti-calpain 2 (1:1000, Abcam), anti-E-cadherin (1:1000, Abcam), anti-GAPDH hFAB rhodamine (Bio-Rad Laboratories), and anti-tubulin hFAB rhodamine (Bio-Rad Laboratories). In addition, anti-mouse or anti-rabbit fluorescent-labeled secondary antibodies (all Bio-Rad Laboratories) were used as secondary antibodies. Blots were scanned and quantified using a ChemiDoc imaging system (Bio-Rad Laboratories). The quantified band intensities were normalized to those of GAPDH or tubulin.

Synthetic elastin peptides (VGVAPG, AGVPGLGVG, GRKRK and TAMRA-AGVPGLGVG) were purchased from Proteogenix. Mouse anti-MMP-2 and anti-uPA antibodies were from Calbiochem. Y27632, blebbistatin, U0126, PD150606, lactose, chondroitin sulphate, nifedipine and EDTA were from Sigma-Aldrich. EGCG was purchased from Enzo Life Sciences. Rabbit anti-Hsp90, anti-cleaved caspase-3, anti-integrin αV, anti-p-ERM, mouse anti-αvβ3 and anti-αvβ5 integrin antibodies were from Ozyme. Rabbit anti-RPSA, anti-MMP-14, anti-calpain1, anti-ROCK2, anti-myosin light chain kinase, mouse anti-RPSA, anti-RhoA, anti-ROCK1 and mouse IgM isotype control antibodies were purchased from Abcam. Rabbit anti-p-LIMK-and goat anti-cofilin and anti-actin were from Santa Cruz. Anti-integrin αvβ3 antibody was purchased from Millipore. Annexin-5 alexa fluor^® 568, CellTrace Calcein Red-Orange, AM and DiOC18³ were from Invitrogen.