Phospho mtor ser2448 d9c2

Olaparib (Selleckchem, AZD2281), rapamycin (LKT Labs, 53123-88-9), and bafilomycin (Sigma-Aldrich, 88899-55-2) were used. The following antibodies were used for the study: Beta-Actin (AC14) (abcam, AB6276, 1:20000 dilution); Atg16L1 (D6D5) (Cell Signaling, 8089 T, 1:1000 dilution); Atg5 (D5F5U) (Cell Signaling, 12994 S, 1:1000 dilution); Atg12 (D88H11) (Cell Signaling, 4180 S, 1:1000 dilution); Beclin-1 (D40C5) (Cell Signaling, 3495 S, 1:1000 dilution); Atg7 (D12B11) (Cell Signaling, 8558 S, 1:1000 dilution); β-Tubulin (D2N5G) (Cell Signaling, 15115 S, 1:1000 dilution); Filamin A (Cell Signaling, 4762 S, 1:1000 dilution); SP1 (Sigma, PLA0307, 1:5000 dilution); anti-phospho-Histone H2A.X (Ser139) (Sigma-Aldrich, JBW301, 1:2000 dilution); LC3 A/B (D3U4C) (Cell Signaling, 12741 S, 1:750 dilution); phospho-mTOR (Ser2448) (D9C2) (Cell Signaling, 5536 T, 1:1000 dilution); SQSTM1/p62 (Cell Signaling, 5114 T, 1:1000 dilution) and (abcam, ab56416, 1:100 dilution); Rad51 (114B4) (abcam, ab213, 1:750 dilution) and BRCA1 (Sigma Millipore, 07-434, 1:1000 dilution), PARP1 (proteintech, 66250, 1:650 dilution) and PAR/pADR (R&D systems, 4335-MC-100, 1:1000 dilution).

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).