Hrp labelled goat anti rabbit igg antibody

Tissue or cells in logarithmic growth phase were added to the cell lysate to extract protein. The protein concentration was determined by BCA method (Sigma). The uploading volume of the sample was adjusted according to the protein quantification result. 30 μg was loaded per well. The protein was separated by SDS-PAGE and transferred onto the PVDF membrane. The membrane was blocked by 50 g/L skim milk powder for 1 h, followed by TBST solution washing for 3 times. Then the samples were incubated with primary antibodies rabbit anti-human PTEN (1:1000, Cat No. 32199, Abcam, Cambridge, UK), Beclin1 (1:1000, Cat No. 62557, Abcam), LC-3 (1:3000, Cat No. 51520, Abcam), hTERT (1:1000, Cat No. 32020, Abcam), and β-actin (1:5000, Cat No. 8227, Abcam) overnight at 4°C on a shaker. After three washes with TBST, HRP-labelled goat anti-rabbit IgG antibody (1:3000 dilution, Cat no. 205718, Abcam, Cambridge, UK) was added for incubation for 1 h, followed by TBST wash for 3 times. The target band was detected by ECL chemiluminescence and recorded using the Bio-image system (Bio-Rad, Hercules, CA, USA). The level of protein expression was normalized with β-actin as an internal reference.

The protein in A549 and A549/DDP and tissues was extracted by Radioimmunoprecipitation assay buffer (RIPA) (Auragene, Changsha, China). Proteins were quantified by bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Proteins were separated on a 10% SDS-PAGE gel and transferred onto nitrocellulose membranes, and then incubated with 5% BSA for 2 h at 25 °C. The membranes were incubated with Anti-CHAF1B antibodies (1:500; ab72520, Abcam); Anti-NCOR2 antibodies (1:500; ab24551, Abcam); Anti-PPP5C antibodies (1:500; sc271612, Santa Cruz) was incubated overnight at 4 °C. Then it was incubated with a secondary HRP-labelled goat anti-rabbit IgG antibody (1:4000; Abcam) for 1 h at room temperature. Immunoassay were performed by ECL combined with Western Blot system (Auragene, Changsha, China). GAPDH (1:4000; ab125247, Abcam) was used as the internal control of Western blotting.