Ab4822

CFs were washed with PBS (0.1 mol/L, pH 7-7.4) three times, lysed in a lysis buffer for 30 min at 4 °C, and then scraped off using a cell scraper before being mixed with a one-quarter volume of 5× loading buffer. The cell protein mixture was then boiled in a 100 °C water bath and cooled to room temperature. The protein concentration was determined using a Bradford protein assay kit (Amresco, M173-KIT). Equal amounts of protein (50 µg) were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The strips were blocked with 5% nonfat milk for 1 h at room temperature. The strips were then incubated overnight with primary antibodies against AAT1 (Sigma, AV48205, 1:500), collagen I (Abcam, ab254113, 1:500), collagen III (Abcam, ab7778, 1:500), α-SMA (Abcam, ab5694, 1:500), β-catenin (Abcam, ab6302, 1:500), p-P38 MAPK (Abcam, ab4822, 1:500), P38 MAPK (Abcam, ab170099, 1:500) and GAPDH (Abcam, ab181602, 1:2000) diluted in PBS with 0.05% Tween 20 at 4 °C. The strips were rinsed using PBS with 0.05% Tween 20 for 30 min, followed by incubation with secondary antibodies for 1 h at room temperature. All protein bands were detected using Amersham ECL Western blotting detection reagents (GE Healthcare, RPN2106) and analyzed with a biological electrophoresis image analysis system.

Cells were grown in 100 mm petri dishes or chamber slides to 80 % confluence. After short washing with PBS, cells were incubated with 2 % (w/v) BSA in calcium binding buffer for 1 h at 37 °C. Afterwards, incubation with 5 µM endotoxin free rS100A12 in calcium binding buffer or with calcium binding buffer alone, as negative control, was performed for 90 min at 37 °C. Cells were lysed for Western blot analysis or fixed using 4 % (w/v) paraformaldehyde for immunocytochemical staining. Western blot analysis of PPARγ synthesis was performed using a monoclonal antibody (abcam191407). Additionally, Western blot analyses of tyrosine kinases Fyn (#4023, cell signalling), pFyn (ab53690, abcam), Lyn (#2796, cell signalling), pLyn (ab33914, abcam) and mitogen-activated kinase p38 (ab4822, abcam) were performed.