Anti mcl 1 s19

S116836 (chemical structure, Fig. 1A) was rationale designed and synthesized in our lab [26 ]. S116836 was dissolved in DMSO at a stock concentration of 20 mM and stored in aliquots at −20°C. U0126 and LY294002 were purchased from Calbiochem (San Diego, CA). Cycloheximide (CHX) was bought from Sigma-Aldrich (St. Louis, MO). MG132 was obtained from EMD Bioscience (Billerica, MA). Antibodies against poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), pro-caspase3, X-linked inhibitor of apoptosis protein (XIAP) and cytochrome c were from BD Biosciences (San Jose, CA). Antibodies against phospho-PDGFRα (Y1018), phospho-Erk1/2 (T202/Y204), Erk1/2, phospho-AKT (S473), AKT, caspase-8, caspase-9, Bax and phospho-Bim (S69) were from Cell Signaling Technology (Beverly, MA). Antibodies against phospho-STAT3 (Y705), STAT3 and PDGFRα were from EMD Millipore Upstate (Billerica, MA). Anti-Mcl-1 (S-19) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bim was from Stressgen Enzo Life Sciences (Plymouth Meeting, Pennsylvania). Anti-Survivin was from Novus Biologicals (Littleton, CO, USA). Anti-active-caspase-3 and Anti-β-actin were from Sigma-Aldrich (St. Louis, MO). Anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G horseradish peroxidase-conjugated antibodies were purchased from Pierce Biotechnology (Los Angeles, CA, USA).

The following antibodies were used: anti-ZO-1/TJP1 (ZO1-1A12) (Fisher Scientific, Cat. No. 33-9100); anti-TAZ (V386) (Cell Signaling Technology, Cat. No. 4883); anti-TEF-1(TEAD1) (Clone 31/TEF-1) (BD Biosciences, Cat. No. 610922); anti-Nrf2 (H-300) (Santa Cruz Biotechnology, Cat. No. sc-13032); anti-MCL1 (S-19) (Santa Cruz Biotechnology, Cat. No: sc-819); and anti-α-tubulin mouse mAb (DM1A) (EMD Millipore Corporation, Cat. No. CP06). Carfilzomib (Cat. No. A-1098) was obtained from Active Biochem; emetine (Cat. No. 32-469-3250MG) was purchased from Fisher Scientific; and homoharringtonine (omacetaxine mepesuccinate) (Cat. No. SML1091) was from Sigma-Aldrich.

The following drugs were purchased: Dinaciclib (SCH727965) for in vitro and in vivo studies (S2768; Selleckchem), lapatinib ditosylate (Tykerb) for in vitro and in vivo studies (M1802; Abmole), neratinib for in vivo studies (M1913; Abmole), A-1210477 (CT-A121; Chemietek), A-1331852 (22963; Cayman Chemicals), tucatinib (HY-16069; Medchem), and ABT-199 (venetoclax) (CT-A199; Chemietek). The antibodies used in this study were as follows: anti-Bak (AB-1 clone for IP) (AM03; EMD Millipore), anti-Bak (3814S; Cell Signaling), anti-Bim (C34C5) (2933S; Cell Signaling), anti–BCL-xL (54H6) (2764S; Cell Signaling), anti–Bcl-2 (D55G8) (Human Specific) (4223S; Cell Signaling), anti-cleaved PARP (Asp214) (D64E10) (5625S; Cell Signaling), anti-GAPDH (6C5) (sc-32233; Santa Cruz), anti–MCL-1 (S-19) (sc-819; Santa Cruz), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (4370S; Cell Signaling), anti-phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) (5364S; Cell Signaling), anti-phospho-Akt (Thr308) (244F9) (4056S; Cell Signaling), anti-phospho-Akt (Ser473) (D9E) (4060S; Cell Signaling), anti-HER2/ErbB2 (29D8) (2165S; Cell Signaling), anti-phospho-HER2/ErbB2 (Tyr1248) (2247S; Cell Signaling), anti-phospho-Rpb1 CTD (Ser 2/5) (4375S; Cell Signaling), Normal Rabbit IgG for IP (sc-2027; Santa Cruz), and Normal Mouse IgG for IP (sc-2025; Santa Cruz).

The antibodies and dilutions used for western blots were as follows: rabbit polyclonal anti-FBXW7 (1:500) and anti-AURKA (1:2,000) from Novus; rabbit polyclonal anti-MCL1 (S-19, 1:1,000), anti-Cyclin B1 (1:1,000), anti-phospho-histone H3 (Ser10, 1:500) and mouse monoclonal anti-PTTG1 (1:1,000) from Santa Cruz; mouse monoclonal anti-β-actin (Ac15, 1:20,000) and rabbit polyclonal anti-c-Myc (1:1,000) from Sigma; rabbit polyclonal anti-cleaved caspase-9 (Asp 315, 1:500), anti-cleaved caspase-3 (Asp 175, 1:500) from Cell Signaling; rabbit polyclonal anti-BubR1 (1:3,000) from Bethyl; mouse monoclonal anti-PARP (1:500) from BD Bioscience; mouse monoclonal anti-Cyclin E (1:500) from Monosan; mouse monoclonal anti-PLK1 (1:10,000) from Millipore, and rat monoclonal anti-HA-peroxidase (50 mU/mL) from Roche. For immunohistochemical analyses, anti- FBXW7 (1:2,000), anti- Cyclin E (1:100), anti- MCL1 (1:1,250), anti-AURKA (1:700), and anti-PLK1 (1:1,000) were used.

For Western blotting analysis, cells were lysed as previously described [63 (link), 64 (link)]. Protein determination was performed by BCA Protein Assay (Thermo Scientific, Rockford, IL). Equal amounts of protein for each sample were migrated in SDS-polyacrylamide gels and blotted onto nitrocellulose filters. The following Abs were used: anti-Mcl-1 (S-19) and anti-Bcl-2 (100) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-AMPKα (Thr172, D79.5E), anti-AMPKα, anti-phospho-STAT3 (Ser727) and anti- STAT3 from Cell Signalling (Danvers, MA); anti-phospho-Akt1/PKBα (Ser473) from Merck Millipore (Darmstadt, Germany); anti AKT/PKBα from BD; anti-tubulin from Sigma-Aldrich. After incubation with anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary Abs (Sigma-Aldrich), specific reactions were revealed with the ECL Lightning detection kit (Perkin Elmer, Waltham, MA). The estimation of the densitometry values of bands was obtained by the ImageQuant TL software (GE Healthcare, Buckinghamshire, UK).