Ab80261

Radio-Immunoprecipitation Assay (RIPA) lysis buffer with cocktail, an effective protease inhibitor (HYK0010, MCE, USA), was used to collect total proteins from myocardial tissues or cells. Protein samples were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto polyvinylidene fluoride (PVDF) membrane (88,518, Thermo, USA). The Primary antibodies including β-actin (sc-47,778, Santa Cruz Biotechnology, 1:1000), cleaved-Caspase 3 (9661, Cell Signaling Technology, 1:1000), cleaved-Caspase 9 (9501, Cell Signaling Technology, 1:1000), BCL2 associated X (Bax) (ab32503, ABcam, 1:1000) or SOX4 (ab80261, ABcam, 1:500) were added to probe specific protein on PVDF membrane. The bands of proteins were detected using Odyssey Infrared Imaging System (Gene Company Limited, Hongkong, China) [20 ].

Cells were lysed by RIPA lysis buffer (Beyotime) to isolate proteins, and then protein concentrations were analyzed using the bicinchoninic acid (BCA) protein assay kit (Takara). Extracted proteins were separated by an SDS-PAGE minigel, and then transferred onto PVDF membranes. After blocking by 5% fat-free milk powder in TBST buffer, membranes were interacted with primary antibodies against SOX4 (ab80261, Abcam, Cambridge, MA, USA), PCNA (ab29, Abcam), Bax (ab32503, Abcam), Cleaved caspase 3 (ab2302, Abcam), Bcl-2 (ab692, Abcam), N-cadherin (ab18203, Abcam), Vimentin (ab92547, Abcam), E-cadherin (ab15148, Abcam), as well as GAPDH (ab181602, Abcam) at 4 °C overnight, followed by incubated with the HRP-conjugated secondary antibody (ab205781, Abcam). Finally, signal visualization was performed by ECL Substrates (Millipore, Billerica, MA, USA).

The total protein extracted from the cell lysate using RIPA buffer (Beyotime, Shanghai, China) was quantified by a BCA assay kit (Beyotime). After 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins were electrotransferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA). Subsequently, the membrane was blocked with 5% skimmed milk and probed with the following primary antibodies from Abcam (Cambridge, UK) rabbit anti-α-SMA (ab32575, 1:2500), rabbit anti-collagen I (ab34710, 1:2500), rabbit anti-SOX4 (ab80261, 1:1500), rabbit anti-DKK1 (ab93017, 1:1500), rabbit anti-β-actin (ab8226, 1:5000), rabbit anti-CD63 (ab134045, 1:1000), rabbit anti-CD81 (ab109201, 1:5000), rabbit anti-CD9 (ab92726, 1:1000), rabbit anti-Alix (ab88388, 1:1000), rabbit anti-TSG101 (ab125011, 1:1000), and rabbit anti-calnexin (ab92573, 1:20000) at 4 °C in the dark. Subsequently, the membrane was re-probed with horseradish peroxidase (HRP)-combined immunoglobulin G (IgG) antibodies (ab6721, 1:5000, Abcam) for 2 h followed by enhanced chemiluminescence (BB-3501, Ameshame, UK) for immunodetection. The protein bands were imaged and analyzed by the BIO-RAD imaging system (California).

Proteins were separated by 10% SDS‐polyacrylamide gel electrophoresis, then transferred to Hybond membranes. We blocked membrances for 1.5 hours at room temperature using 5% fat‐free. Then, primary antibodies purchased from Abcam, including SOX4 (ab80261, 1:1000) and β‐actin (ab8227, 1:5000) were incubated overnight at 4°C. And then, the membrane was washed three times with TBST and the secondary antibodies were added for 1 hour at room temperature. Finally, protein was visualized using an enhanced chemiluminescence system and visualized after X‐ray film exposure.