Ab14824

RIPA lysis buffer (Beyotime) and BCA protein assay kit (Beyotime) were utilized for total protein extraction and quantification, respectively. Then, equal amount of protein (20 μg/lane) was segregated by SDS-PAGE gel and then shifted into PVDF membrane (Solarbio). After blocked in 5% non-fat milk for 1 h, the membrane was probed with specific primary antibodies at 4 °C overnight, and subsequently probed with HRP-conjugated secondary antibody (1:20,000, ab205718 or ab205719, Abcam, Cambridge, MA, USA) for 2 h. The blots were exposed to enhanced BeyoECL Moon (Beyotime), and the bands density was assessed via Image J software (NIH, Bethesda, MD, USA). The primary antibodies obtained from Abcam included anti-B-cell lymphoma-2 antibody (Bcl-2; 1:1000, ab32124), anti-BCL2-associated X protein antibody (anti-Bax; 1:1000, ab32503), anti-matrix metalloproteinase 2 (MMP2) (1:1000, ab86607) antibody, anti-MMP9 (1:1000, ab137867) antibody, anti-PPM1A (1:500, ab14824) antibody, and β-actin antibody (1:5000; ab6276). Relative protein expression was normalized by internal reference β-actin.

For IHC, sections were toasted at 60°C for 4 hours before deparaffinization and dehydration. Slices were then soaked in sodium citrate solution and heated at 60°C for 4 hours to make antigen retrieval. Subsequently, sections were incubated with primary Abs at 4°C overnight and treated for secondary Abs for 20 minutes the next day. The positive signal was visualized with DAB reagents (ZSGB-BIO) and slices were counterstained with hematoxylin. For immunofluorescence (IF), samples were incubated with a fluorescent secondary Ab for 40 minutes at room temperature and DAPI was used for nuclear staining. The primary Abs employed in the current study include Col2a1 (Abcam, catalog ab34712; 1:500, IF), PPM1A (Bioss, catalog bs-4162R; 1:300, IHC; and Abcam, catalog ab14824; 1:100, IF), MMP-13 (Abcam, catalog ab39012; 1:300, IHC), Adamts-5 (Bioss, catalog bs-3573R; 1:200, IHC), and p-SMAD2 (Ser465 and Ser467) (Thermo Fisher Scientific, catalog 44-244G; 1:300, IHC/IF). Quantitative analysis was performed using ImageJ software.