Bs 1115r

Western blots were performed as previously described [20 (link)]. The primary antibodies used were anti-MyHC (B103, 0.5 μg/mL, DHSB, Iowa City, IA, USA), anti-MYOD (ABP53067, 1:500, Abbkine, Wuhan, China), anti-MYH1A (F59, 0.5 μg/mL, DHSB, Iowa City, IA, USA), anti-MYH7B (S58, 0.5 μg/mL, DHSB, Iowa City, IA, USA), anti-NAMPT (bs-0272R, 1:500, Bioss, Beijing, China), anti-p-AMPK (ABN-PAB12602, 1:2000, Abnova, Taipei City, Taiwan, China), anti-AMPK (bs-1115R, 1:500, Bioss, Beijing, China), anti-PGC1α (66369–1-Ig, 1:5000, Proteintech, IL, USA), and anti-β-Tubulin (A01030, 1:10,000, Abbkine, Wuhan, China). ProteinFind Goat Anti-Mouse IgG (H + L), HRP Conjugate (HS201–01, 1:1000, TransGen, Beijing, China) and ProteinFind Goat Anti-Rabbit IgG (H + L), HRP Conjugate (HS101–01, 1:500, TransGen, Beijing, China) were used as a secondary antibody.
Immunofluorescence were performed using anti-Desmin (bs-1026R, 1:100, Bioss, Beijing, China) and anti-MyHC (B103, 2.5 μg/mL, DHSB, Iowa City, IA, USA), as previously described [20 (link)]. A fluorescence microscope (DMi8; Leica, Germany) was used to capture three randomly selected fields to visualize the area labeled with anti-MyHC.

The phosphofructokinase 1 (PFK1) (sc-31711) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while the phosphofructokinase 2 (PFK2) (bs-3528R), carnitine palmitoyltransferase-1A (CPT-1A) (bs-2047R), AMP-activated protein kinase (AMPK) (bs-1115R), β-actin (bs-10966R), phosphorylated PFK2 at Ser467 (bs-3331R), and phosphorylated pyruvate kinase M2 (p-PKM2) at Tyr105 (bs-3334R) antibodies were all purchased from Bioss Biotechnology (Beijing, China). The antibodies against PKM2 (15822–1-AP), hexokinase 2 (HK2) (22029–1-AP), α-ketoglutarate dehydrogenase (OGDH) (15212–1-AP), citrate synthase (CS) (16131–1-AP), isocitrate dehydrogenase 2 (IDH2) (15932–1-AP), lactate dehydrogenase B (LDHB) (14824–1-AP), and pyruvate dehydrogenase E1(PDHE1) (18068–1-AP) were all purchased from Proteintech Group (Chicago, IL, USA). The phosphorylated AMPK (Tyr172, 2531) antibody was acquired from Cell Signaling Technology (Beverly, MA, USA). The macrophage migration inhibitory factor (MIF) antibody was produced in our laboratory. Hematoxylin (AR11180–1) and eosin (AR11180–2) staining solutions were purchased from Boster Bioengineering Co. Ltd. (Wuhan, China), while the RIPA protein buffer and BCA protein assay kit (P0010) were procured from Beyotime Biotechnology (Shanghai, China). ECL-plus reagent (AR1111) was obtained from Bioss Biotechnology.