Western blot analysis
C2C12 myotubes (1X106) were incubated at different
concentrations of AdipoAI and AdipoRON for 90 minutes. The cells were
then washed with ice-cold PBS. The pellet was lysed using ice-cold 1X
NUPAGE LDS lysis buffer (Invitrogen) approximately 200ul of the sample
buffer were added to each sample the cells were vortex briefly and
centrifuge at 8,000rpm for 5 mins, the supernatant was collected and
heated at 70°C for 10min. The proteins were than loaded in 4.12%
bis-tris gel using MES running buffer (Invitrogen) at 100V for 2 hours
(Qiu et al., 2021; Wu et al., 2022), proteins were then transferred to
0.2 um polyvinylidene difluoride membrane using transfer buffer
(Tris-base, glycine,10% SDS, methanol). Membrane was blocked using 3%
bovine serum albumin (BSA) in TBST for 1 hour followed by incubation
with specific antibody (p-AMPK, APPL1, PGAC-y) overnight. After primary
antibody the membrane were than washed 3 times with TBST for 10minutes
and incubated with HRB labeled secondary antibody containing 3% BSA for
1h at room temperature, after incubation the membrane were washed 3
times with TBST and were visualized using ECL reagent (Ahuja et al.,
2020).