Full range rainbow prestained protein standards

MACS-isolated FAPs from tibialis anterior muscle were lysed at 4°C using RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, protease and phosphatase inhibitors (Calbiochem and Roche, respectively). Cell lysates were cleared by centrifugation at 16,000 g for 10 min, and protein concentration was determined using a BCA assay (Thermo Fisher Scientific). The cell lysate was denatured at 100°C for 5 min in 6X SDS-PAGE loading buffer. 5–10 µg of cell lysates were resolved on 4–15% TGX gradient gels (Bio-Rad Laboratories) and transferred to nitrocellulose membrane (Amersham). Membranes were blocked with 5% non-fat dried milk in TBS with 0.1% Tween, and then incubated with primary antibody overnight at 4°C. Primary antibodies used were mouse anti-GLI3 (1:1,000; gift of Suzie Scales, Genentech) and mouse anti-αTUB (1:5000; Sigma #T5168). After washing in TBS with 0.1% Tween, membranes were incubated in HRP-conjugated secondary antibody for 2hr at room temperature. Blots were developed with Western lightning enhanced chemiluminescence (PerkinElmer), and molecular weights were determined using full-range rainbow pre-stained protein standards (GE Healthcare).