Sds page loading buffer

Monoclonal antibody against p17 was prepared by our lab. CQ, antibodies against LC3 or β-actin and horse radish peroxidase-labeled secondary antibodies were purchased from Sigma-Aldrich (Shanghai, China). RIPA lysis buffer, PMSF and SDS-PAGE loading buffer were purchased from Beyotime Biotechnology (Shanghai, China). Chemiluminescent substrate was purchased from Thermo Fisher Scientific (Shanghai, China).

RIPA lysis buffer (Beyotime) was used according to the manufacturer’s instructions to extract the total protein from coelomic fluid. The protein concentration was measured using a BCA protein assay kit (Beyotime). The protein samples were then mixed with SDS-PAGE loading buffer (Beyotime) and denaturized at 100 °C for 8 min. The samples were separated on 4–15% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk in 0.1% Tween 20 in TBS buffer (TBST) for 2 h, incubated in primary antibody in TBST (1:500 dilution) overnight at 4 °C, washed 3 times in TBST for 36 min, incubated in secondary antibody in TBST (horseradish peroxidase-labeled goat anti-rat IgG [H+L], Beyotime; 1:2000 dilution) for 2 h at 37 °C, and washed 3 times in TBST for 45 min. Finally, the membranes were incubated with developer and photographed using a chemiluminescence imaging system. The gray scale values of MFN2, DRP1 and ACTB proteins blotting were measured by using ImageJ software (National Institutes of Health, Baltimore, MD, USA). The ratio of their gray scale values were used to analyze the relative expression of the MFN2 and DRP1 proteins. Three samples were used for each experiment.

Western blotting was performed as described previously (11 (link)). Briefly, A549 cells seeded in 24-well plates were transfected with miRNA or siRNA or pcDNA-AIVR. Twenty-four hours later, the cells were washed twice with cold PBS and lysed in RIPA lysis buffer (Beyotime, China) containing protease inhibitors (Merck, Germany) and phenylmethylsulfonyl fluoride (PMSF) (Biosharp, China). The protein extracts were denatured at 100°C in SDS-PAGE loading buffer (Beyotime) for 10 minutes. Equal amounts of protein were run on SDS-PAGE. Proteins were transferred onto a nitrocellulose filter membrane (GE Healthcare, USA) and incubated with primary antibody. After being washed with PBS with Tween (PBST), the blots were incubated with secondary antibody and visualized by using an Odyssey CLx infrared imaging system (Li-Cor, USA).