C myc

Western blotting was performed using a SDS-PAGE Electrophoresis System according to the previous description [16 (link), 17 (link)] with antibodies specific for C-myc (Cell Signaling Technology, Beverly, MA, USA), CDK4 (Cell Signaling Technology), CDK6(Cell Signaling Technology), CyclinD1(Cell Signaling Technology), Rb (Cell Signaling Technology), p-Rb (Cell Signaling Technology), Caspase3 (Immunoway, USA), Cleaved Caspase3 (Cell Signaling Technology), Snail (Proteintech, USA), Slug (Proteintech), E-cadherin (Cell Signaling Technology), N-cadherin (Cell Signaling Technology), PI3K (Abclonal Technology), p-PI3K(Cell Signaling Technology), AKT(Cell Signaling Technology), p-AKT(Cell Signaling Technology), PTTG1(Cell Signaling Technology), β-Tublin (Cell Signaling Technology) and β-actin (Proteintech).Signals were detected using enhanced chemiluminescence reagents (Millipore, Schwalbach/Ts., Germany).

Human colon cancer cell lines (DLD-1, HCT-15, and HCT-116) at 50–60% confluence were harvested after an overnight treatment with bazedoxifene or DMSO, and then lysed in cold RIPA lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail. The lysates were subjected to 10% or 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with a 1:1000 dilution of specific primary antibody and 1:10,000 HRP-conjugated secondary antibody. Primary antibodies against phosphorylated STAT3 (Tyr705, p-STAT3^Y705), IL-6, BCL-XL, c-MYC, survivin, cyclin D1, STAT3, AKT, ERK, phospho-specific extracellular signal-regulated kinase (ERK) 1/2 (threonine 202/Tyrosine 204), phosphorylated-AKT (Ser473), GAPDH and secondary antibodies were all from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against IL-11, IL-11Rα and IL-6R were from Abcam (Cambridge, MA, USA). Membranes were analyzed using enhanced chemiluminescence plus reagents and scanned with the Storm Scanner (Amersham Pharmacia Biotech Inc., Piscataway, NJ).

Cells were harvested and lysed in RIPA buffer (Cell Signalling Technology, Danvers, MA), and the resultant proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PolyScreen membranes (NEN, Boston, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and probed with antibodies targeting the following proteins: β-catenin (Cell Signalling Technology), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), AXIN2, c-Myc, STAT3, cyclin B1, Gab1, c-caspase-3, p-ERK (Cell Signalling Technology), HA, β-actin, and γ-tubulin (Santa Cruz Biotechnology). Primary antibodies were detected with HRP-conjugated anti-mouse or anti-rabbit antibodies, as appropriate, that were subjected to enhanced chemiluminescence (Amersham, Buckinghamshire, UK).

Taselisib, also called GDC-0032, was generated at Genentech, Inc. (South San Francisco, CA). Letrozole was obtained from US Biological. Antibodies used include phospho-AKT^Ser473, AKT, phospho-PRAS40^Thr246, phospho-S6^Ser235/236, phospho-S6^Ser240/242, S6, phospho-ERK^{Thr202/Tyr204}, ERK, phospho-ERα^Ser118, phospho-ERα^Ser167, cleaved PARP, p110α, phospho-p70S6K^Thr389, PR, cyclin E, phospho-mTOR^Ser2448, IGF1R, BRCA1, c-Myc, CAV1, HER2 and cyclin D1 obtained from Cell Signaling (Danvers, MA). Antibodies for ERα and ERβ were obtained from Santa Cruz biotechnology (Santa Cruz, CA) and a βActin antibody was obtained from Sigma (St. Louis, MO).

Western blot was performed according to the manufacturer’s protocol. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gels followed by transfer to PVDF membranes. Membranes were probed with primary antibodies overnight and then washed and incubated with horseradish peroxidase (HRP)-conjugated-secondary antibodies. Detection was performed by the enhanced chemical luminescence (ECL) method. The primary antibodies of NCOR2 (ab24551; 1:1000) was purchased from Abcam. CRBN (NBP1-91810; 1:1,000) antibody was purchased from Novus Biologicals. c-Myc (#5605; 1:1,000), IKAROS (#9034; 1:1,000), MTA1 (#5647; 1:1,000), MBD3 (#14540; 1:1,000), HDAC1 (#5356; 1:1,000), and HDAC2 (#5113; 1:1,000), antibodies were purchased from Cell Signaling Technology, Inc.

MG132(52801ES08) and CHX (40325ES03) were purchased from Yeason (Shanghai, China). The following antibodies were used for western blotting: USP16 (A5861; Abclonal), c-Myc (GTX103436; GeneTex), β-actin (sc-47,778; Santa Cruz Biotechnology). Antibodies used for immunohistochemistry: USP16 (HPA021140; Sigma-Aldrich), Ki67 (sc-15,402; Santa Cruz), c-Myc (#ab32072, Abcam). Antibodies used for immunoprecipitation and immunofluorescence: Flag (#30503ES60, Yeason), USP16 (HPA021140; Sigma-Aldrich), c-Myc (13987S, Cell Signaling Technology).

Antibodies against NUAK1 (#4458S), LKB1 (#3050S), p-LKB1-Ser428 (#3482S), LC3B (#2775), and c-myc (#5605) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-tubulin antibody (#T5168), Anti-actin antibody (#A2066), anti-rabbit FITC secondary antibody (# F9887), HRP-conjugated antibodies against mouse IgG (NA931V), and rabbit IgG (NA934V), and 4′,6-diamidino-2-phenylindole were purchased from Sigma (St. Lewis, MO, USA). Chloroquine (#C-6628), MG132 (#M8699), and methylcellulose (#M0512) were also obtained from Sigma. Anti-fibronectin (#ab2413) was purchased from Abcam (Cambridge, MA, USA). Antibody against L1CAM (#SIG-3911) was obtained from Biolegend (San Diego, CA, USA). Anti-USP9X (A301-350A) was purchased from Bethyl Laboratories (Montgomery, TX, USA). Alexa Fluor phalloidin and plasma human fibronectin (#PHE0023) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). WZ4003 (#5177) was purchased from Tocris Bioscience.