Anti gapdh antibody

Cells and tissues was lysed on ice in RIPA (KeyGEN Biotech) buffers for 20 min and then centrifuged at 12,000× g, 15 min. Protein concentration was measured by BCA kit (Thermo Scientific, Pittsburgh, PA, USA). Proteins at 20 μg were loaded to 10% SDS-polyacrylamide gel electrophoresis and were transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Anti-EN2 antibody (ab28731, Abcam), anti-EN2 antibody (MAB2600, R&D), anti-EN2 antibody (ab45867, Abcam), anti-GAPDH antibody and anti-DDK antibody (OriGene Technologies, Rockville, MD, USA) were used as the primary antibody for incubation at 4 °C overnight and the secondary antibodies labeled with horseradish peroxidase (HRP) as used for incubation at room temperature for 1 h. ECL chemiluminescent system was performed for the detection.

Transforming growth factor-β1 (cat #: T1654), UA (cat #: 03240595), and protein A agarose (cat #: P1406) were purchased from Sigma–Aldrich (St. Louis, MO, United States). Matrigel (cat #: 354263) was purchased from Corning Incorporated (New York, NY, United States). Puromycin (cat #: REVG1001) was purchased from GENECHEM (Shanghai, China). The anti-NOX4 (cat #: ab109225), anti-RhoA (cat #: ab187027), anti-ROCK1 (cat #: ab205829), anti-α-SMA (cat #: ab32575), anti-Collagen-I (cat #: ab6308), anti-p67^phox (cat #: ab109366), anti-Rac1 (cat #: ab33186), anti-TIMP1 (cat #: ab61224), anti-MMP1 (cat #: ab137332), anti-MMP2 (cat #: ab37150), anti-MMP9 (cat #: 119906), and anti-F-actin (cat #: ab205) antibodies were purchased from Abcam (Cambridge, MA, United States). The anti-GAPDH antibody was purchased from OriGene (cat #: TA802519) (Rockville, MD, United States). The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).