Cleaved parp d214

The antibodies used and commercial sources are as follows: cyclin B1, CHK1, EGFR, AKT, pAKT^S473, JAK2, pJAK2^Y1007/1008, STAT3, pSTAT3^Y705, c-Myc, Met. IGF-1 R, cleaved PARP^D214, cleaved caspase-3^D175, cleaved caspase-7^D198, cleaved caspase-9^D330, Src, pSrc ^Y418, PI3K (p110β), Erk, pErk ^T202/Y204, S6, pS6^S235/236 (Cell Signaling, Danvers, MA, USA); CDK1, Ki-67 (Abcam, Cambridge, MA, USA); and pCDK1(Thr14/Tyr15), β-actin (Sigma-Aldrich, St. Louis, MO, USA); p-EGFR^Y1173 (Life Technologies (Invitrogen)). The drugs, ganetespib, dasatinib, alisertib, everolimus, and cisplatin, were purchased from Selleck Chemicals Inc. (Houston, TX, USA). The siRNAs siGL2 (targeting firefly luciferase GL2), siEGFR1, and siEGFR2 (targeting human EGFR) were synthesized by Shanghai GenePharma Co. (Shanghai, China) and transfected with Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA). The siRNAs used are as follows: siGL2, (F) 5′- UCGAAGUAUUCCGCGUACG-3′ and (R) 5′-CGUACGCGGAAUACUUCGA-3′ siEGFR1, (F) 5′- GGAGAUAAGUGAUGGAGAUTT-3′ and (R) 5′- AUCUCCAUCACUUAUCUCCTT-3′ siEGFR2, (F) 5′-CCCAGAAGGUGAGAAAGUUTT-3′ and (R) 5′- AACUUUCUCACCUUCUGGGTT-3′.

Cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) NP40, 0.5% (v/v) Na-deoxycholate, 0.1% (v/v) SDS] with 1 tablet/10 ml Complete Mini EDTA-free protease inhibitors (Roche). SDS-PAGE and immunoblots were performed using standard protocols.
Antibodies against the indicated proteins were used as follows – HIF2α (PA1-16510, Thermo Scientific), Cezanne (custom antibody, Eurogentec), β-actin (3700, Cell Signaling), PHD3 (A300-327A, Bethyl Labs), BNIP3 (ab10433, Abcam), Glut-1 (53519, Anaspec), p52 (05-361, Merck Millipore), E2F1 (3742, Cell Signaling), SP1 (07-645, Upstate-Millipore), cyclin D1 (DCS6, Cell Signaling), cyclin E (HE12, Cell Signaling), cyclin A (C-19, Santa Cruz), GFP (2956, Cell Signaling), cleaved PARP (D214) (9541, Cell Signaling), c-Myc (9E10, Sigma), phosphorylated Chk1 at S345 (2341, Cell Signaling).