Ab133448

Western blot was conducted as per our previous reports^{6 (link),25 (link),48 (link)}. Briefly, blots were first incubated with antibodies to the following proteins: Nrg4 (1:1,000 dilution, Thermo Fisher, PA5-102641), P-IKKβ (Ser176/180) (1:1,000 dilution, CST, 2697), IKKβ (1:1,000 dilution, CST, 2684), P-p65 (Ser468) (1:1,000 dilution, CST, 3039), p65 (1:1,000 dilution, CST, 8242), P-IκBα (Ser32) (1:1,000 dilution, CST, 2859), IκBα (1:1,000 dilution, CST, 9242), P-ERK (Thr202/Tyr204) (1:1,000 dilution, CST, 4376), ERK (1:1,000 dilution, CST, 4695), P-Akt (Ser473) (1:1,000 dilution, CST, 4051), Akt (1:1,000 dilution, CST, 4691), AMPK (1:1,000 dilution, Abcam, ab32047), P-AMPK (Thr183 + Thr172) (1:1,000 dilution, Abcam, ab133448), ErbB4 (1:1,000 dilution, Abcam, E200), MMP2 (1:1,000 dilution, Abcam, ab92536), MMP9 (1:1,000 dilution, Abcam, ab76003) and GAPDH (1:3,000 dilution, CST, 5174). Binding of the primary antibody was detected by incubating membranes with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:2,000 dilution, Abcam, ab205719).

The extracted protein lysates from ischemic myocardial tissues and cardiomyocytes were used for Western blot analysis as described previously [27 (link)]. The lysates were normalized to equal amounts of protein, and 10 μg protein from cell lysates or 30 μg protein from tissue lysates were separated by 10% or 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane, and then sealed with 5% nonfat milk in TBST for 3 h. Membranes were incubated with the following primary antibodies: p-AMPK (1:2000, #ab133448, Abcam), AMPK (1:1000, #ab80039, Abcam), PGC-1α (1:1000, #ab191838, Abcam), SIRT3 (1:1000, #ab264041, Abcam), SOD2 (1:5000, #ab13533, Abcam), Bax (1:2000, #ab182733, Abcam), B-cell-lymphoma protein 2 (Bcl-2) (1:2000, #ab182858, Abcam), caspase3 (1:2000, #ab184787, Abcam), cleaved-caspase3 (1:5000, #ab214430, Abcam), and β-tubulin (1:20,000, #66240-1-lg, Proteintech) at 4 °C overnight. Proteins were then probed with specific HRP-conjugated secondary antibodies for 30 min at room temperature. Finally, the protein bands were obtained using the ChemiDoc Touch Imaging System (Chemi Doc, Bio-Rad, Hercules, CA, USA), and the relative intensity of the bands was quantified using Image Lab software.

Tissues were homogenized with 5 volumes of radioimmunoprecipitation assay (RIPA) buffer (Solarbio^® Life Sciences), and the supernatants were fractionated by SDS–PAGE. The proteins were quantified with a BCA protein quantification kit (Solarbio^® Life Sciences), transferred to PVDF membranes (Bio–Rad), and blocked with 5% blocking solution for Western blotting (Roche). The membranes were then exposed to an anti-eNOS antibody (EPR19296) (ab199956, Abcam; dilution: 1/1000), an anti-AMPK alpha 1 + AMPK alpha 2 antibody (EPR19549) (ab207442, Abcam; dilution: 1/1000), a recombinant anti-AMPK alpha 1 (phospho-T183) + AMPK alpha 2 (phospho-T172) antibody (EPR5683) (ab133448, Abcam; dilution: 1/5000); an anti-PGC1 alpha rabbit pAb (#A11971, ABclonal; dilution: 1/1000), an anti-mTOR (phospho-S2448) antibody (EPR426[2 (link)]) (ab109268, Abcam; dilution: 1/5000), an anti-mTOR (phospho-S2481) antibody (EPR427[N]) (ab137133, Abcam; dilution: 1/5000), an anti-adiponectin antibody (EPR17019) (ab181281, Abcam; dilution: 1/1000), and an anti-GAPDH antibody (EPR16891) loading control (ab181602, Abcam; dilution: 1/5000). Immunodetection was conducted using a goat anti-rabbit IgG H&L (HRP) (ab6721, Abcam; dilution: 1/3000) secondary antibody and an enhanced chemiluminescence device (Bio–Rad gel imager).

Proteins were extracted from tissues and cells with cell lysis buffer, boiled for 5 min, and stored at -80°C until use. Samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred to polyvinylidene fluoride (PVDF) membranes blocked with 5% nonfat dry milk in TBS-Tween (blocking buffer) for 1 h. PVDF membranes were probed at 4°C overnight in blocking buffer with the following primary antibodies: MSTN (ab201954, Abcam, USA), p-Smad2 (ab280888, Abcam, USA), p-Smad3 (ab52903, Abcam, USA), Smad2 + Smad3 (ab202445, Abcam, USA), GLUT1 (A11727, ABclone, China), GLUT4 (A7637, ABclone, China), p-AMPKα1+α2 (ab133448, Abcam, USA), G6PD (ab993, Abcam, USA), TKL (1 : 1000, ab181235, Abcam, USA), RPI (ab137629, Abcam, USA), phosphoserine (ab9332, Abcam, USA), phosphotyrosine (ab10321, Abcam, US), p-AKT (ab38449, Abcam, USA), p-P38 (ab31828, Abcam, USA), and α-tubulin (11224-AP, Proteinch, China). Membranes were washed three times and incubated with secondary antibody diluted 1 : 5000 in blocking buffer. Finally, protein expression was detected and recorded.

Western blotting was performed with the SDS-PAGE electrophoresis system. Adherent cells or adipose tissue extracts were prepared and transferred to PVDF membranes. The following primary antibodies were used: anti-GAPDH (ap0063, Bioworld), anti-Col15α1 (ab150463, Abcam), anti-Caspase-9 (ab32539, Abcam), anti-Cleaved Caspase-9 (bs7070, Bioworld), anti-Caspase-3 (Bs6428, Bioworld), anti-Cleaved Caspase-3 (bs7004, Bioworld), anti-Bcl2 (bs1511, Bioworld), anti-Bax (ab32503, Abcam), anti-AMPK (ab32047, Abcam), anti-pAMPK (ab133448, Abcam), anti-mTOR (ab87540, Abcam), anti-pmTORC1^Ser2448 (ab109268, Abcam), anti-Akt (ab8805, Abcam), anti-pAkt^Ser473 (ab18206, Abcam), anti-S6K1 (ab32529, Abcam), anti-pERK1/2 (ab201015, Abcam), anti-ERK1/2 (ab17942, Abcam), anti-pS6K1^Thr389 (ab2571, Abcam), anti-Collagen I (ab34710, Abcam), anti-Collagen VI (ab6588, Abcam), anti-Fibronectin (ab2413, Abcam), anti-MMP2 (ab92536, Abcam), anti-MMP9 (ab38898, Abcam), anti-TIMP1 (WL02342, Wanleibio), anti-TIMP2 (ab180630, Abcam), anti-FGFR1 (ab31324, Abcam), anti-pFGFR1^{Tyr653/Tyr654} (GTX133526, GeneTex), anti-TGFβ1 (WL03092, Wanleibio). Horseradish peroxidase anti-rabbit or anti-goat (Sigma–Aldrich) were used as secondary antibodies.

MLO-Y4 cells were homogenized in lysis buffer, and protein concentration was quantified using the Bradford assay. After separation through 15% SDS-PAGE and transferred onto PVDF membranes, cell lysate was subjected to immunoprecipitation with specific antibodies for 1 h at RT, followed by immunoblotting according to the kit’s protocols (Roche, Basel, Switzerland). The primary antibodies used for immunostaining were: rabbit anti-JNK1 (1:1000, Abcam, ab199380), rabbit anti-p-JNK1 ab47337, rabbit anti-AMPK (1:1000, Abcam, ab32382), rabbit anti-p-AMPK (1:1000, Abcam, ab133448), rabbit anti-Beclin 1(1:1000, Abcam, ab207612), rabbit anti-Bcl-2 (1:1000, Abcam, ab182858), rabbit anti-LC3 (1:1000, Abcam, ab128025), rabbit anti-Cleaved Caspase-3 (1:1,000, Abcam, ab32042), rabbit anti-Cleaved PARP (1:1000, Abcam, ab32064), rabbit anti-SOD1 (1:1000, Abcam, ab179843), rabbit anti-SOD2 (1:1000, Abcam, ab74231), rabbit anti-Actin (1:3000, Abcam, ab8227). The secondary antibodies used for immunostaining were: mouse HRP (1:2000, Abcam, ab6728), rabbit HRP (1:2000, Abcam, ab6721).

Total protein was extracted from lung tissue and quantified through the BCA assay. Each sample containing 40 μg of mixed loading buffer was boiled at 100 °C for 15 min. The sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with QuickBlock™ blocking buffer (P0235, Beyotime) at 25 °C for 10 min and then rinsed with Western wash buffer (P0023C, Beyotime) for 5 min 3 times. Anti-rabbit p-AMPK (dilution: 1:500, ab133448, Abcam, Cambridge, UK), anti-rabbit AMPK (dilution: 1:1000, ab32047, Abcam, Cambridge, UK), anti-rabbit cleaved-caspase-1 (dilution: 1:500, ab179515, Abcam, Cambridge, UK), anti-rabbit cleaved-caspase-3 (dilution: 1:500, ab32351, Abcam, Cambridge, UK) and GAPDH (dilution 1:1000, K106389P, Solarbio) were used for overnight incubation of PVDF membranes at 4 ℃. After rising with Western washing buffer 3 times, the membranes were incubated with goat anti-rabbit secondary antibody (dilution 1:1000, A0562, Beyotime) at 25 ℃ for 1 h. After washing 3 times with Western washing buffer, protein bands were detected using BeyoECL Moon (P0018, Beyotime). The ratio between the gray value of the target protein and the GAPDH bands (internal reference) was calculated using ImageJ.