P bad

Western blot analysis was performed as preciously described. Lysates of treated cells or tumor samples were collected and lysed in radioimmunoprecipitation assay buffer. Equal amounts of proteins were resolved on SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. Specific primary antibodies were used for the following molecules : IĸBα(Cabiochem, San Diego, CA, USA), p65, Iĸĸα, Iĸĸβ, AKT, p-AKT, MEK, p-MEK, ERK, p-ERK, p38, caspase-10, caspase-1, cyclinA, cyclinB, PUMA, BID, CIAP, Bad, p-Bad, PKC (Cell signaling Technology), JNK2/1, p-JNK, TSP1, Bcl-2, Bcl-xL, Bax, APAF1, Fas, caspase-8, Mrg1, cyclinD, p53, MMP1 (Santa Cruze Biotechnology, CA), caspase-9, caspase-3 (BioLegend). β-Actin (Santa Cruze Biotechnology, CA) was used to assess the equal loading. The phosphorylated protein controls obtained from blots that had been stripped and re-probed for the same sample lane. Sixty microgram of cell protein extract was loaded per lane. CS-710 Calibrated Imaging Densitometer (Bio-Rad) was utilized for densitometric quantification.

MM cells were harvested and lysed as mentioned previously.^{9 (link)} Lysates were then measured for protein quantification, boiled (100 °C for 5 min), run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis 4–15% gradient gels at 110 V (Bio-Rad, Hercules, CA, USA), transferred to nitrocellulose membranes and immunoblotted with antibodies to the following: pCHK2 (Thr68), pCHK1 (Ser345), pH2AX (Ser139), PIM1, PIM2, PIM3, Caspase-3, PARP, BAD, pBAD (Ser112), p21 Waf1/Cip1, MDR-1, pYAP1 (Ser127), YAP1, ABL1 and GAPDH (Cell Signaling, Beverly, MA, USA), which was used as a loading control. All antibodies were diluted 1:1000 in 5% bovine serum albumin/TBST (TBS plus Tween-20).

Ganetespib was purchased from Medkoo Biosciences, Inc. (Chapel Hill, NC). Lapatinib was purchased from LC Laboratories (Woburn, MA). Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies specific to cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl-2, ErbB2, phospho-ErbB2 (pErbB2), Akt, pAkt, Erk2, pErk1/2, mTOR, pmTOR, Cyclin B1, Src, pSrc, Bad, pBad, GSK3, and pGSK3 were purchased from Cell Signaling Technology (Danvers, MA). Primary antibodies against CDK1, Cyclin D1, p27, caspase 8, caspase 9, HSP90, HSP70, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary mouse and rabbit antibodies were purchased from Thermo Scientific (Rockford, IL).

All chemicals and reagents used in this study were purchased from Merck (Merck KGaA, Darmstadt, Germany). The antibodies used in this study were purchased from different manufactures including CD90 and CD45 (BD Biosciences, East Rutherford, NJ, USA); p-Akt, p-IGF1R, PI3K, p-PI3K, Akt, p-Bad, Bcl-xL, Fas-L, and FADD (Cell Signaling Technology, Danvers, MA, USA); b-actin, AMPKa, p-AMPKa, and Sirt1 (Santa Cruz Biotechnology, Dallas, TX, USA); and Caspase-8 and TGF-b (Promega-US).

Lapatinib was purchased from LC Laboratories (Woburn, MA). MG132, CHX, RNase A, and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO). LY294002 and PD98059 were purchased from Alexis Biochemicals (San Diego, CA). EGF was purchased from Peprotech (Rocky Hill, NJ). Rapamycin and antibodies against pErbB2 (Tyr1221/1222), ErbB2, pEGFR (Tyr1068), EGFR, pmTOR (Ser2448), mTOR, pAkt (Ser473), Akt, pBad, Bad, and PARP were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against CIP2A, c-Myc, pErk1/2 (Thr202/Tyr204), Erk2, Caspase-3, cleaved Caspase-3, cyclin D1, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Western analyses were performed as described25 (link). For each experiment, lysates from a cell line grown with or without serum were run on the same gel and all gels were run at the same time. Two gels were transferred to a single membrane. If more than one membrane was used for an experiment, a single solution for each primary and secondary antibody pair was made and divided between the two membranes that were subsequently hybridized in separate trays. Antibodies used included pAKT(S473) (product #4060), pAKT(T308) (product #2965); pan-AKT (product #2920 and 4691), pPRAS40 (product #2997), PRAS40 (product # 2610), pS6(S235) (product #2211), pS6(S240) (product #5364) S6 (product #2317 and 2217), pFOXO1/FOXO3a (product #9464), FOXO3a (product #2497), pGSK3α/β (product #9331), GSK3α/β (product #5676), pBAD (product #4366) and BAD (product #9239) from Cell Signaling Technologies (Danvers, MA, USA) or β-actin (product #A5441) from Sigma-Aldrich (St. Louis, MO, USA). Membranes were scanned and intensities calculated using a semi-automated infrared imaging system (Odyssey scanner and Image Studio Lite software, LiCor BioSciences Inc., Lincoln, NE, USA). Histograms show ratios of infrared signal intensities of indicated antibodies. For experiments using two membranes, histograms of infrared signal ratios graphed by membrane are shown in Supplementary Fig. 8.