Hrp goat anti rabbit secondary antibodies

After lysis with RIPA lysis buffer (Beyotime, Shanghai, China), the Hepa1-6 cells and liver tissue were centrifuged at 14,000 × g at 4 °C for 15 min. Then, 10% sodium dodecyl sulfate–polyAcrylamide gel electrophoresis (SDS-PAGE; Sinopharm, Shanghai, China) was used to resolve extracted proteins and polyvinylidene difluoride (PVDF) membranes (Millipore, Shanghai, China) was used to blot them. The membrane was blocked with 5% skimmed milk in Tris-buffered saline (room temperature, 1 h), followed by culturing with primary antibodies against LC3 (Cell Signaling Technology, Danvers, MA, USA; 1:1000 dilution), Sequestosome-1(p62) (Cell Signaling Technology, Danvers, MA, USA; 1:3000), LAMP2 (Abcam, Cambridge, MA, USA; 1:1000), mTOR (Cell Signaling Technology, Danvers, MA, USA; 1:1000) and SIRT1 (Abcam, Cambridge, MA, USA; 1:1000) at 4 °C overnight. Next, HRP-Goat anti-rabbit secondary antibodies (ASPEN, Wuhan, Hubei, China; 1:10,000) were cultured with it for 2 h at room temperature. GAPDH (Abcam, Cambridge, MA, USA; 1:10,000) was applied as a loading control. Chemiluminescence (ECL) substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used to see the protein bands and the ImageJ software was utilized to semi-quantify them.

The cells were lysed in RIPA buffer (Aspen Biotechnology, Wuhan, China) supplemented with Protease Inhibitor Cocktail (Roche, NJ, USA) for 5 min. The concentration of total protein was determined using BCA protein assay kit (Aspen Biotechnology). Total protein samples (40 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking in 5% skim milk at room temperature for 1 hr, the PVDF membrane was incubated with primary antibodies at 4° C overnight. The PVDF membrane was washed three times with tris-buffered saline and tween 20 (TBST) then incubated with HRP-Goat anti Rabbit secondary antibodies (1:10000, Aspen) for 2 hr at room temperature. The blots were visualized with an enhanced chemiluminescence (ECL) kit (Aspen). β-actin was utilized a loading control. The primary antibodies for western blot were: HMGA1 (1:1000, Abcam, Cambridge, MA, USA), Bax (1:2000, Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1:1000, Abcam), cleaved caspase-3 (1:1000, Abcam), β-actin (Abcam, 1:1000).