Chemiluminescence reagent

Cellular proteins were extracted using RIPA lysis buffer (G2002, Servicebio, Wuhan, China), and protein concentrations were determined using a BCA kit (G2026, Servicebio, Wuhan, China). Subsequently, proteins were separated through electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, Burlington, Massachusetts, USA). After blocking with 5% non-fat milk, the membranes were incubated with specific primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at 25 °C. Chemiluminescence reagent (G2014, Servicebio, Wuhan, China) was employed for signal detection using the Clinx imaging system (Shanghai, China). Primary antibodies against P4HA1 (1:1000, 12658-1-AP) and β-actin (ACTB, 1:1000, 20536-1-AP) were purchased from Proteintech (Wuhan, China), and against TET2 (1:1000, A5682), FBP1 (1:1000, A11664), P4HA2 (1:1000, A22150), P4HA3 (1:1000, A13767), P4HB (1:1000, A19239) from AbClonal (Wuhan, China). The primary antibody for hypoxia-inducible factor-1α (HIF-1α, GTX127309) was purchased from Gene Tex (Irvine, CA, USA). The primary antibody for Flag (1:1000, MA1-91878) was purchased from Thermo Scientific (USA). Gastrocnemius muscle tissue proteins were extracted using the same procedure followed by homogenization.

A volume of 20 μL of lysed samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. The membrane was blocked using 3% skim milk before incubation with primary antibodies, specifically mouse anti-CD63 (1:500; Servicebio, China). Next, the membrane was incubated with goat anti-mouse IgG antibodies conjugated to horseradish peroxidase (1:5,000; Servicebio, China) and visualized using a chemiluminescence reagent (Servicebio, China). The immunoreactivity was detected on a Bio-Rad ChemiDoc MP (Bio-Rad, USA).