Gapdh

Cell lysates were created using radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl pH 7.6) containing a protease/phosphatase inhibitor (Cell Signaling Technology, Danvers, MA, USA). Protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, Illinois, USA). Approximately 30 μg of total protein from each sample was boiled for 10 min, separated by SDS-PAGE, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with primary antibodies against anti-PTEN (Abcam, Cambridge, MA, USA, 1 : 500), anti-OTUD3 (Abcam, 1 : 100), anti-pSer473-AKT (Cell Signaling Technology, 1 : 1,000), and GAPDH (Abgent, San Diego, CA, USA; 1 : 1,000). After washing and incubating with secondary antibodies (Sigma Aldrich, Darmstadt, Germany, 1 : 10000), the blots were visualized with an ECL reagent (Millipore).

Whole cell lysates were prepared as described previously [19 (link)]. Equal amounts (30 μg) of total proteins were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, followed by immunoblotting analyses with specific antibodies including antibodies against RNF6 (Thermo Fisher), Flag, Myc, HA (Medical & Biological Laboratories, Tokyo, Japan), ERα, Bcl-xL, Bim-1 (Cell Signaling Technologies., Ltd), or GAPDH (Abgent, Suzhou, China). Anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems.

Whole cell lysates were prepared as described previously [11 (link)]. After proteins were then transferred to polyvinylidene difluoride membranes, the blots were then probed with antibodies including monoclonal PARP, Caspase-3, p-AKT(S473), p-AKT(T308), AKT, p-mTOR(S2448), Raptor, p-P70S6K, P70S6K, p-4E-BP1(S65), 4E-BP1 (all were purchased from Cell Signaling Technology, Inc.). GAPDH was purchased from Abgent. β-actin, anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems.

Protein was extracted with ProteoJET Mammalian Cell Lysis Reagent (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitor. Protein samples were treated with Dual Color Protein Loading Buffer (Thermo Fisher Scientific) containing reducing agent at 100 °C for 5 min, resolved on 10% Tris-HCl polyacrylamide gels, and transferred to a polyvinylidene fluoride membrane. Overnight incubation (4 °C) of the primary antibody was followed by HRP-conjugated anti-rabbit (1 : 1000; Novus Biologicals, Littleton, CO, USA) or anti-mouse antibody (1 : 1000; Novus Biologicals) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Antibody dilution was as follows: SPAG5, 1 : 300 (Sigma-Aldrich); mTOR, 1 : 1000 (Cell Signaling, Danvers, MA, USA); S6K, 1 : 1000 (Cell Signaling); phosphorylated (pi)-S6K (Thr 389), 1 : 1000 (Cell Signaling); 4E-BP1, 1 : 500 (Cell Signaling); pi-4E-BP1 (Thr 37/46), 1 : 1000 (Cell Signaling); and GAPDH, 1 : 1000 (Abgent, San Diego, CA, USA).

For the western blot analysis, the cells were treated with DAC and ATRA for 48 h and then collected. Total protein from the cell line was extracted using cell lysis buffer for western blot anaylsis containing phenylmethanesulfonyl fluoride (Beyotime) according to the manufacturer’s instructions. The proteins were separated by 10% SDS polyacrylamide gel and transferred to nitrocellulose membranes. Following blocking with 5% skimmed milk, the membranes were incubated with an appropriate dilution of the rabbit anti-human polyclonal primary antibodies, anti-p16 (1:100), -RAR-β (1:100) and -GAPDH (1:1,000) (ABGENT, San Diego, CA, USA), followed by incubation with the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ABGENT), according to manufacturer’s instructions. The signals were displayed using an automatic film processor (SX435-T; Taixing Suxing Co., Ltd., Taixing, China). The signal intensity of the proteins was normalized against GAPDH using Quantity One software (Bio-Rad, Hercules, CA, USA).

Western blotting analysis was performed following the standard protocols (BioRad). Total proteins from tissues and cells were extracted with RIPA lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor and phosphatase inhibitor. Antibodies used in this study were CIDEA (NBP1-76950, Novus), GAPDH (AM1020B, Abgent), p21 (#2947, Cell Signaling Technology), CylinD1 (#2926, Cell Signaling Technology), CDK4 (#12790, Cell Signaling Technology), p-JNK (Thr183/Tyr185) (#4668, Cell Signaling Technology), Bad (#9239, Cell Signaling Technology), Caspase9 (#9508, Cell Signaling Technology), and PARP (#9542, Cell Signaling Technology).

Whole cell lysates were prepared for immunoblotting as described previously [32 (link), 33 (link)]. Specific primary antibodies against PARP, Caspase 3, MCL1, XIAP, p-STAT3(Tyr705), STAT3, p-STAT1(Tyr701), STAT1, p-STAT5(Tyr694), STAT5, JAK2, p-Src(Ser17), c-Src, p-P38(Thr180/Tyr182), P38, p-ERK1/2(Thr202/Tyr204), ERK1/2, p-mTOR(Ser2448) and mTOR were purchased from Cell Signaling Technology (Danvers, MA); a specific primary antibody against p-JAK2(Tyr1007/Tyr1008) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-Flag and anti-Myc antibodies were purchased from Medical & Biological Laboratories (Tokyo, Japan). GAPDH and β-actin were purchased from Abgent (Suzhou, China). Anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems (Minneapolis, MN).