Actin 4967

MCF7 breast carcinoma cells were obtained from ATCC (Manassas, VA, USA). RPMI culture medium, fetal bovine serum and SuperScript reverse transcriptase were obtained from Invitrogen (Carisbad, CA, USA). Antibodies for nSMase2 (H195), Sp1 (PEP2) and Sp3 (D20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Chk1 (2360), Chk2 (2662), ATM (2873), ATR (2790), p53 (9282) and actin (4967) were from Cell Signaling Technologies (Beverly, MA, USA). The inhibitors AZD7762 (S1532) and MK-8776 (S2735) were from Selleck Chem (Boston, MA, USA). Go6976 (2253) was purchased from Tocris Biosciences (Bristol, UK). Bisindolylmaleimide I (13298) was obtained from Cayman Chemicals (Ann Arbor, MI, USA). siRNA for TP53 (s607) and CREB3L1 (s40546) were from Life Technologies (Grand Island, NY, USA). All other siRNAs, CHEK1 (SI00024570), CHEK2 (SI00095305), ATM (SI00000840), ATR (SI00023107), Sp1 (SI150983) and Sp3 (SI0004788) were from Qiagen (Hiden, Germany). Porcine brain sphingomyelin and phosphatidylserine were from Avanti Polar Lipids (Alabaster, AL, USA). Doxorubicin, actinomycin D and, unless indicated otherwise, all other chemicals were obtained from Sigma (St. Louis, MO, USA).

Reagents for Western blotting were obtained from Bio-Rad (Hercules, CA, USA). Proteins were electrophoretically separated on a sodium dodecyl sulfate (SDS)-polyacrylamide gel, followed by transfer to nitrocellulose membranes. The transformation of pcDNA6.2-GW/EmGFP-mi-INHA into S. Typhimurium was assessed by monitoring the co-cistronic expression of EmGFP using a monoclonal anti-GFP antibody (ab1218, Abcam). The expression of bacterial chaperone protein DnaK was probed for simultaneously using an anti-DnaK antibody (ab69617, Abcam) as the internal loading control. The other proteins in mammalian cancer cells were detected using the following primary anti-bodies: INHA (ab81234) and Bcl-2 (ab59348) from Abcam; Bcl-xL (2764) and actin (4967) from Cell Signaling (Beverly, MA, USA). Secondary antibodies were horseradish peroxidase labeled anti-mouse or anti-rabbit antibodies. Binding was detected by chemiluminescence signals, according to the manufacturer’s instructions (Roche, Basel, Switzerland).