Protein Quantification and Analysis by Western Blot

RIPA protein lysates and Western blot analysis were performed as previously described [13 (link)]. For immunoblotting, 30–60 μg of protein were used. Primary antibodies were used as follows: mouse anti-TGFBR2 (sc-17799; Santa Cruz, Dallas, USA; 1:500, 4°C, overnight); mouse anti-ß-Actin (clone 4; MP Biomedicals, Solon, USA; 1:20,000, RT, 30 min); rabbit anti-GDF-15 (HPA011191; Sigma, Taufkirchen Germany; 1:500, 4°C, overnight); rabbit anti-phospho-Smad2 and rabbit anti-Smad2 (Ser465/467) and (86F7); Cell Signaling, Danvers, USA; 1:1000, 4°C, overnight). Blots were incubated for 1 h at RT with secondary antibodies: sheep anti-mouse-IgG HRP (1:5000; GE-Healthcare, Munich, Germany) or goat anti-rabbit-IgG HRP (1:2500; Promega, Madison, USA). Detection was either performed by standard procedure using Amersham Hyperfilm ECL or ChemiDoc MP System (Bio-Rad Laboratories GmbH, Munich, Germany).