P0018as

The cellular proteins were extracted form skeletal muscle cells and lysed by RIPA lysate (R0010, Solarbio, Peking, China). Equal amounts of protein were electrophoresed on sodium lauryl sulfate polyacrylamide gel (SDS-PAGE) with a loading of 20 μL per well. The protein was then transferred to an activated PVDF membrane (FFP26, Beyotime, Shanghai, China) and blocked with 5% skim milk powder (P0216, Beyotime, Shanghai, China) for 2 h. Appropriately diluted primary antibodies against Beclin-1 (Abcam, Cat# ab223372), p62 (Abcam, Cat# ab101266), p-mTOR (Abcam, Cat# ab109268), mTOR (Abcam, Cat# ab2732), p-AKT^Ser473 (Abcam, Cat# ab18206), p-IRS1^Ser616 (CST, Cat# 2386S), IRS (CST, Cat# 2382S), AKT (CST, Cat# 2938S), LC3 (Affinity, Cat# AF5402) and β-actin (Abcam, Cat# ab8227) were added and incubated at 4 °C overnight. After washing the membrane, the corresponding secondary antibody (Cat# A0208 and A0216, Beyotime, Shanghai, China) was added for incubation. Chemiluminescence detection was then carried out using an enhanced chemiluminescence reagent (P0018AS, Beyotime, Shanghai, China). After development and fixing treatment, the film was photographed by a gel imaging analysis system (UniCel Dxl800, Beckman Coulter, Pasadena, CA, USA).

Total proteins were extracted by using RIPA protein lysate (89900, Invitrogen, USA). 30 μg of total protein was separated by 10% SDS-PAGE. Primary antibodies against E-cadherin (1 : 1000, AF0131, Affinity Biosciences, USA), α-SMA (1 : 1000, XBT-1, Affinity Biosciences, USA), ZEB2 (1 : 1000, AF5278, Affinity Biosciences, USA), GAPDH (NCI5079, Good Here, China), CD9 (1 : 1000, bs-2489R, Bioss, China), CD63 (1 : 1000, GTX41877, GeneTex, USA), and CD81 (1 : 800, GTX41794, GeneTex, USA) were employed to incubate with activated membrane overnight at 4°C. Then, the bands were detected by incubation with HRP conjugate (1 : 5000, BA1054, Boster, China) secondary antibody at room temperature for 2 h. Chemiluminescence detection was conducted using an enhanced chemiluminescence reagent (P0018AS, Beyotime, China), and the images were observed by a gel imaging analysis system (UniCel DxI800, Beckman Coulter, USA).