Goat anti mouse igg

Total protein was extracted from the HeLa and HeLa/DDP cells. The protein concentration was measured by bicinchoninic acid (BCA) and 30 μg protein Page: 8 was loaded into a polyacrylamide gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After wet transfer, the nitrocellulose membrane was blocked with 5% skim milk for 40 min, followed by being incubated overnight at 4 °C with the primary antibody specific for P16 (Proteintech, US), P-gp (Abcam, US), pERK1/2 (Cell Signaling Technology, US), total ERK1/2 (Cell Signaling Technology, US) and β-actin (Chinese fir golden bridge company, China). The next day, the film was washed with Tris Buffered Saline Tween (TBST) buffer 15 min for three times and then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (CWBIO, China) or goat anti-mouse IgG (TransGen, Beijing, China) secondary antibody at room temperature for 1 h. The film was washed again with TBST buffer and finally stained with an enhanced chemiluminescent agent and exposed to a gel imaging system (Chemi DOCtm XRS, Bio-Rad, Beijing, China).

After the total proteins of cells in each group were isolated with total protein extraction kit (Transgen, DE101-01), protein samples were treated with SDS-PAGE protein loading buffer (Beyotime, P0015). Then the cell proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, which were transferred to a polyvinylidene fluoride membrane (Millipore), washed with TBST buffer (Solarbio, T1081). The membrane was sealed with 5% skim milk for 1h, and incubated with primary antibody at 4 °C overnight, and the second antibody incubated at 37 °C for 1h. Then, the samples were washed 3 times with TBST buffer (Solarbio, T1081), and ECL luminescence solution (Biosharp, BL520A) was added. The protein bands were observed using a chemiluminescence gel imaging system (Bio-RAD, USA). The used antibodies are as follows: GAPDH (Transgen, HC301-01), HSPA5 (Proteintect, 11587-1-AP), LC3B (Abcam, ab192890), P62 (BOSTER, M00300-1), Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536T), mTOR (7C10) (Cell Signaling Technology, 2983T), Phospho-Akt (Ser473) (D9E) (Cell Signaling Technology, 4060S), Akt (pan) (C67E7) (Cell Signaling Technology, 4691S), Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (ab275018), Goat Anti-mouse IgG (Transgen, HS201-01), Goat anti-rabbit IgG (Transgen, HS101-01).