Af5384

Retinal tissue was extracted and lysed with RIPA (Solarbio, R0010, Beijing, China) lysis buffer for 30 min on ice. After centrifuging the lysate at 12,000 rpm for 15 min at 4 °C, the supernatant was collected. The protein concentration was determined using the BCA protein assay kit (Solarbio, PC0020, Beijing, China). The same amount of protein was loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were immersed in a blocking solution of 50 g L⁻¹ nonfat dry milk for 2 h. The corresponding primary antibodies (1/1000, LC3B, 18725-1-AP, Proteintech, USA; 1/1000, P62, AF5384, AffinitY, USA) were added to the membrane overnight at 4 °C. Then, the membranes were incubated with the corresponding secondary antibody (1/1000, MDL, MD912565, Hebei, China) for 1 h at 37 °C on a shaker. Finally, immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Solarbio, PE0010, Beijing, China) and detected using an automated image analysis system (170-8280, ChemiDoc MP Chemiluminescence Imaging System; Bio-Rad, CA, USA). Statistical analysis was performed with β-actin as an internal reference.

Th total protein extracts were prepared from 10 to 15 mg of the deep-frozen heart tissue. To maintain the extract integrity and function, we used Complete Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA), Phosphatase Inhibitor Cocktail II (ab201113, Abcam, Waltham, MA, USA), PMSF (1 mM), EGTA (1 mM), and EDTA (1 mM). The proteins were isolated using a 1xRIPA buffer (ab156034, Abcam, Waltham, MA, USA). The concentration of the protein was determined by the Lowry method [15 (link)]. The samples (30–50 µg) were diluted in Laemmli buffer, separated by 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Wilmington, USA) [12 (link)]. The relative levels of the detected proteins were visualized using a C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, Nebraska, USA) and normalized to the appropriate loading control. The antibodies used: (ab56788) anti-DRP1 (1:1000), (ab119685) anti-OPA (1:1000), (ab124773) anti-Mitofusin2 (1:1000), (ab54481) anti-PGC1a (1:1000), (cs2132) anti-Parkin (1:1000), (Affinity Biosciences, DF7742) anti-PINK1 (1:250), (Affinity Biosciences, AF5384) anti-SQSTM1/p62 (1:250), (Affinity Biosciences, DF8163) anti-BNIP3L (1:250), and (cs12741) anti-LC3A/B (1:1500). The anti-GAPDH (ab181602) and anti-tubulin (1:2000) (ab4074) antibodies were used as a loading control.

Immunohistochemical analysis was performed as previously described.²⁹ Briefly, the solid tumors were removed, fixed using 10% formaldehyde, and embedded in paraffin. The sections were blocked using 0.3% hydrogen peroxide at room temperature for 20 min to remove endogenous peroxidase activity. Next, nonspecific protein binding was blocked by incubation the sections with 2% bovine serum albumin and with primary antibody against Nur77 (12235AD; Proteintech), p62 (AF5384; Affinity), CD68 (ab955; Abcam), and Ki67 (ab15580; Abcam) overnight at 4°C; then, the secondary antibody was added, carried with horseradish peroxidase, and developed according to the manufacturer's protocol (IHC staining module; Beijing Zhongshan Biotechnology). The sections were counterstained using hematoxylin, and the immunohistochemical staining results were observed under a light microscope (Olympus IX71).

Immunofluorescence analysis of H9c2 cells with LBAG treatment was employed to assess the levels of autophagy-related protein, p62. In short, the H9c2 cells were fixed, washed, and then incubated with the diluted p62 antibody (1 : 200, AF5384, Affinity Biosciences Ltd., USA) at 4°C overnight. Ultimately, the images were captured using a microscope.

The primary antibodies used in this study included MMP2 (40994, Cell Signaling Technology, Boston, USA), MMP9 (13667, CST, USA), E-cadherin (14472, CST, USA), β-cadherin (8480, CST, USA), vimentin (5741, CST, USA), Twist1 (ab50887, Abcam, Cambridge, USA), LC3 II/I (12741S, CST USA), p62 (AF5384, Affinity, USA), PI3K (AF6241, Affinity, USA), p-PI3K (ab32089, Abcam, Cambridge, USA), Akt (4691, CST, USA), p-Akt (5536, CST, USA), p-mTOR (9271, CST, USA), mTOR (2983, CST, USA), GSK3β (5676, CST, USA), p-GSK3β (9322, CST, USA), PARP (ab32138, Abcam, USA), cleaved-caspase 3 (9664, CST, USA), caspase 3 (9662, CST, USA), cleaved-caspase 8 (8592, CST, USA), caspase 8 (ab108333, Abcam, USA), ATG5 (12994, Abcam, USA), and tubulin (5335, CST, USA).

Total protein of the left ventricular tissue of rats and H9c2 cells with LBAG treatment was extracted using lysis buffer containing PMSF on ice. The protein levels were estimated with a BCA method (Thermo Fisher Scientific). Equivalent protein (20 μg) was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). After being blocked, the membranes were then incubated with primary antibodies including Bax (1 : 2000, AF0120, Affinity), Bcl-2 (1 : 1000, AF6139, Affinity), cleaved-caspase 3 (1 : 1000, AF7022, Affinity), p62 (1 : 2000, AF5384, Affinity), LC3 (1 : 1000, AF5402, Affinity), Beclin 1 (1 : 1000, AF5128, Affinity), PI3K (1 : 1000, AF6241, Affinity), phospho-PI3K (1 : 1000, AF3241, Affinity), AKT (1 : 1000, AF6264, Affinity), phospho-pan-AKT1/2/3 (Ser473) (1 : 1000, AF0016, Affinity), mTOR (1 : 2000, AF6308, Affinity), phospho-mTOR (Ser2448) (1 : 1000, AF3308, Affinity), and β-actin (1 : 5000, AF7018, Affinity) overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies (1 : 5000, Kangchen, China) for 1.5 h at room temperature. Protein bands were visualized using an enhanced chemiluminescent (ECL) kit (Beyotime, China). The image intensity was analyzed using the Image J software.