Ab109217

The cultured cells or tissues were harvested and used to extract total proteins by RIPA lysis (KCD-M1013, Cronda). The concentration of the total protein from each treatment was detected using Bicinchoninic acid assay (BL521A, Biosharp). Then equal protein was used to load on 12% Sodium dodecyl sulfate polyacrylamide gel electropheresis. The proteins were transferred to Polyvinylidene fluoride membranes after separation. Subsequently, the membranes were sealed by 5% non-fat milk for 2 hours and incubated with primary antibodies anti-IRF2 (1 : 1000, abcam, ab124744), anti-CD6 (1 : 2000, abcam, ab109217), anti-CD9 (1 : 5000, abcam, ab236630), anti-TSG101 (1 : 5000, abcam, ab125011), anti-caspase 3 (1 : 2000, abcam, ab32351), and anti-actin (1 : 2000, Serivicebio, GB12001) for one night at 4°C. The membrane was cultured by secondary antibody (1 : 4,000, BL003A, biosharp) for 1 hour at 37°C. Finally, the protein band was imaged by enhanced chemiluminescence (ECL) in a darkroom. Image J was used to detect the relative grey density.

Total protein was extracted from the serum or heart tissues using RIPA lysis buffer (P0013, Beyotime, Shanghai, China) according to the manufacturer’s instructions^{41 (link)}. Twenty grams of protein aliquots derived from each experimental samples, were assessed with the BCA protein assay kit (70-PQ0012, MultiSciences, China). They were boiled at 100 °C for 5 min, separated using 10–12% SDS-PAGE electrophoresis and transferred onto PVDF membranes. The membranes were then blocked with 5% lipid-free milk/TBST buffer for 2 h at room temperature, and then incubated with anti-PDPK1 antibody (ab234064, 1:1000, Abcam, UK), with anti-HIF1a (20960-1-AP, 1:200, Proteintech, USA), with anti-VEGF (19003-1-AP, 1:1000, Proteintech, USA), with anti-VEGFR (26415-1-AP, 1:1000, Proteintech, USA), with anti-CD6 (ab109217, 1:1000, Abcam, UK), with anti-CD9 (ab92726, 1:500, Abcam, UK), with anti-TSG101(ab125011, 1:1000, Abcam, UK) and with anti-GAPDH (ab32391, 1:1000, Abcam, Cambridge, UK) at 37 °C for 2 h, respectively. Then, all blotting membranes were subsequently incubated with secondary anti-mouse IgG antibodies (ab205719, 1:20000, Abcam, Cambridge, UK) for 1–2 h. All the immuno-complexes were finally detected using ECL after washing with TBST and analyzed using the Image-Pro Plus 6.0 software.