Ab267787

Total cellular protein was extracted using lysis buffer, and protein levels were quantified using a BCA kit (ASPEN Biotechnology, Wuhan, China). Proteins were separated on a 10% SDS-PAGE before being probed with the appropriate primary antibodies for CD9, CD63 TSG101, PI3Kγ (#ab154598, Abcam, Cambridge, UK, 1:500), PIK3R1 (#4257, CST, MA, US, 1:1000), P-Akt (#4060, CST, MA, US, 1:1000), IL-17 (#sc-374218, Santa Cruz, Texas, US, 1:500), PTEN (#ab267787, Abcam, Cambridge, UK, 1:2000) and Bcl-2 (#ab182858, Abcam, Cambridge, UK, 1:1000) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were then probed with secondary antibodies, and protein was identified using a LiDE110 scanner (Canon, Japan). GAPDH (#ab37168, Abcam, Cambridge, UK, 1:10,000) was used for normalization, and the AlphaEaseFC software was used to measure protein band density.

Total proteins were extracted from the left ventricles of murine hearts or cultured cells using the RIPA lysis buffer as previously described.³¹, ³² Next, 20 μg total proteins were separated by 10% SDS‐PAGE, transferred onto PVDF membranes, blocked with 5% skim milk at room temperature and incubated with indicating primary antibodies at 4°C overnight. On the second day, the membranes were probed with horse radish peroxidase (HRP)‐conjugated secondary antibodies and visualized with an electrochemiluminescence reagent. The bands were analysed using an Image Lab software and normalized to GAPDH. The following antibodies were used at a 1:1000 dilution: anti‐NRF2 (#ab92946, Abcam), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, #ab8245, Abcam), anti‐phosphorylated AKT (p‐AKT, #4060, Cell Signaling Technology), anti‐total AKT (t‐AKT, #4685, Cell Signaling Technology) and anti‐PTEN (#ab267787, Abcam).

Total cellular protein was extracted using RIPA lysis buffer (P0013B, Beyotime, China). The protein content of the samples was determined using the BCA protein assay kit (BL521A, Biosharp, China). The proteins were mixed with SDS-PAGE loading buffer (MB2479, Meilunbio, China) and separated by gel electrophoresis onto PVDF membranes. Subsequently, the membranes were blocked using 5% nonfat milk. Primary antibodies including HDAC3 (1:1000, ab137704, Abcam, UK), PTEN (1:1000, ab267787, Abcam, UK), PIK3 (1:1000, 4257, Cell Signaling Technology, USA), p-PIK3 (1:1000, 4228, Cell Signaling Technology, USA), AKT (1:1000, 9272, Cell Signaling Technology, USA), p-AKT (1:2000, 4060, Cell Signaling Technology, USA), and β-actin (1:1000, ab8227, Abcam, UK) were incubated with the membranes overnight at 4 °C. Then, the membranes were incubated with the respective secondary antibodies. The target protein bands were visualized using a chemiluminescence imaging system (Chemiscope6100, CLiNX, China). β-actin was used as an internal reference to normalize the protein expression levels.

Immunohistochemical staining was used to detect the expression of PTEN. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned with a 3 μm section. After incubation with primary and secondary antibody, the sections were stained with DAB reagent. Primary antibody: anti-PTEN (ab267787, Abcam, CA), secondary antibody: goat anti-rabbit IgG (Biosci, Wuhan China). All slices were photographed at a magnification of × 400.