Anti β actin antibody

The following reagents were used in this study: Pronase E (PE), Nycodenz, UA and diphenyleneiodonium chloride (DPI) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Type IV collagenase and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). DNase I was purchased from Generay Biotech Co., Ltd. (Shanghai, China). Recombinant human TGF-β1 was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Thermo Fisher Scientific, Inc. Antibodies against NOX2/gp91^phox, p22^phox, desmin, Rac1, sterol-4-alpha-methyl oxidase (Smo), Gli family zinc finger 2 (Gli2), and α-SMA were purchased from Abcam (Cambridge, MA, USA). Antibodies against p67^phox and sonic hedgehog (Shh) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). An anti-β-actin antibody and goat anti-rabbit secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). RNA simple Total RNA kit, Real Master Mix, and FastQuant RT kit were purchased from Tiangen Biotechnology Co., Ltd. (Beijing, China). Primers were synthesized by Beijing Genomics Institute (Beijing, China).

Tissue samples were washed in pre-cooled phosphate-buffered saline three times and lysed in a nondenaturing tissue lysis buffer containing protease inhibitors to extract total proteins. Cell lysate proteins were resolved by polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% normal fetal bovine serum at room temperature for 2 h, incubated with mouse anti-FAT10 monoclonal antibody (dilution, 1:400; Santa Cruz Biotechnology) or anti-β-actin antibody (dilution, 1:200; Zhongshan Golden Bridge, Beijing, China) at 4°C overnight, washed with Tween Tris Base Buffer Solution (commonly known as TTBS) three times, and then incubated with a horseradish-peroxidase-labeled secondary antibody (dilution, 1:400; Zhongshan Golden Bridge) at room temperature for 2 h. After the membranes were washed three times with TTBS, the proteins were detected by enhanced chemiluminescence. Images were obtained using the EC3 Imaging System, and the bands were semiquantitatively analyzed using ImageJ software to calculate the relative expression of FAT10 to β-actin. The experiment was repeated at least three times, with mean values calculated for further analysis.