Actin 60008 1 ig

Total proteins were extracted by sonication in RIPA (Radioimmunoprecipitation assay buffer) buffer containing protease and phosphatase inhibitors, and the protein concentration was measured using the BCA (Bicinchonic Acid) kit (Thermo Fisher Scientific, Waltham, MA, USA); 40 μg of protein was loaded on 7% PAA (Polyacrylamide) gel. After electrophoresis, they were transferred to a nitrocellulose membrane (Amersham BioSciences, Little Chalfont, England, UK), blocked with 5% milk and incubated with primary antibodies overnight. Antibodies used for detection were as follows: rabbit anti-GLI1 (Cell Signaling Technology, V812, 1:200, Danvers, MA, USA), mouse anti-GLI2 (Santa Cruz Biotechnology, sc-271786, 1:200, Dallas, TX, USA), rabbit anti-GLI3 (GeneTex GTX104362, 1:1000, Irvine, CA, USA). Actin (60008-1-Ig, ProteinTech, 1:4000, Rosemont, IL, USA) was used as a loading control. After washing in TBST (Tris-Buffered Saline, 0.1% Tween^® 20 Detergent), secondary antibodies HRP (Horseradish Peroxidase)-conjugated anti-rabbit (BD Pharmingen, 554021, 1:6000, San Jose, CA, USA) and anti-mouse (BD Pharmingen, 554002, 1:8000, San Jose, CA, USA) were applied for 1h at room temperature, washed, and visualized using SuperWest Signal Pico and Femto reagents (Thermo Fisher Scientific, Waltham, MA, USA) on Uvitec Image Alliance 4.7 instrument (UVItec, Cambridge, England, UK).

The whole cell lysates were harvested and subjected to SDS-PAGE (10% separation gel) and transferred to a polyvinylidene difluoride (PVDF) membrane. Primary antibodies against the following proteins were used: HMGB1 (10829-1-AP; Proteintech), TGFBI (10188-1-AP; Proteintech), GAPDH (10494-1-AP; Proteintech), and actin (60008-1-Ig; Proteintech). Horseradish peroxidase–conjugated secondary antibodies were used (ZSGB-BIO). Signals were detected using an ECL (enhanced chemiluminescence) kit (Millipore).