Myc sc 40

Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.
The following chemical compounds and recombinant proteins were commercially obtained: EPZ015666 (S7748) and GSK591 (S8111) from Selleck; Human IL‐6 (96‐200‐06‐20) from Peprotech and TGF‐β (TGFB1‐100) from StemRD.

Cells were lysed on ice for 10 min using NETN lysis buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, 0.5% Nonidet P-40, 50 mM β-glycerophosphate, 10 mM NaF, and 1 mM Na₃VO₄) containing a protease inhibitor cocktail (535140, Millipore, Burlington, MA, USA). After centrifugation at 12,000× g for 5 min, the supernatant was saved as a crude cell extract. This was boiled in Laemmli buffer and loaded onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel. Western blotting was performed according to a standard protocol. The following antibodies were used for Western blotting: FLAG (F3165, Sigma, St. Louis, MO, USA), HA (MMS-101R, Covance, Princeton, NJ, USA), Myc (sc-40, Santa Cruz Biotechnology, Dallas, TX, USA), HIF-1α (610958, BD Biosciences, Franklin Lakes, NJ, USA), PPM1G (ab70794, Abcam, Cambridge, UK, USA), and β-actin (4970, Cell Signaling, Danvers, MA, USA).

The following antibodies were used in this study for western blots and immunoprecipitation assays: anti-CHIP (rabbit C3B6; Cell Signaling Technology, Danvers, Massachusetts, USA), MYC (sc-40; Santa Cruz Biotechnology, CA, USA), FLAG (mouse F3165; Sigma-Aldrich, St. Louis, MO, USA), FLAG (rabbit; Sigma-Aldrich), HA (mouse sc-7392; Santa Cruz Biotechnology, CA, USA), PPARγ (mouse sc-7273X, Santa Cruz Biotechnology), PPARγ (rabbit sc-7196X; Santa Cruz Biotechnology), aP2 (goat sc-18661; Santa Cruz Biotechnology), C/EBPα(rabbit sc-61X; Santa Cruz Biotechnology), β-actin (A5316; Sigma-Aldrich), MG132 (M-1157, A.G.Scientific, San Diego, CA, USA), and geldanamycin (9843 S, Cell Signaling Technology). Pepstatin A (P5318), aprotinin (A1153), phenylmethanesulfonylfluoride (PMSF; P7626), leupeptin (L2884), dimethyl sulfoxide (DMSO; D8418), N-ethylmaleimide (NEM; E3876), dexamethasone (D1756), 3-isobutyl-1-methylxanthine (IBMX; I5879), and Oil Red O (O0625) were purchased from Sigma-Aldrich (St. Louis, MO, USA), while insulin (11 376 497 001) was purchased from Roche (Mannheim, Germany).

Protein fractions were separated by SDS-PAGE (8% to 15% depending on the experiment) and transferred onto nitrocellulose. Membranes were blocked with 1% BSA in PBS-Tween 0.1% (PBS-T) for 1 h, followed by incubation with primary antibodies in blocking buffer overnight at 4°C. Membranes were washed three times with PBS-T for 10 min and then incubated with secondary antibodies conjugated to horseradish peroxidase in blocking buffer for 1 h. Membranes were washed four times with PBS-T for 15 min, and signals were detected by enhanced chemiluminescence (Santa Cruz sc-2048 or Biorad Clarity 1705061). Antibodies to SUMO1 (FL-101, 1:1000 dilution), SUMO2 (FL-103, 1:2000 dilution), Ubc9 (H-81, 1:500 dilution), His (H-15, 1:500 dilution), p53 (DO-1, 1:5000 dilution), myc (sc-40; 1:2000 dilution) and actin (C-11, 1:2000 dilution) were from Santa Cruz. Antibodies to PML (A301-167A, 1:2000 dilution), GST (A190-122A; 1:10,000) and FLAG (S190-102, 1:10000 dilution) were from Bethyl. Anti-FLAG (F1804, 1:10000 dilution) was from SIGMA and anti-ICP0 (H1A027, 1:5000 dilution) was from Virusys. RNF4 antibody (a kind gift from Ronald T. Hay; [109 (link)]) and was used at 1:5000 dilution.

Immunoblotting was performed as previously described [26 (link)]. Briefly, inputs or immunoprecipitates were separated by SDS-PAGE and blotted using antibodies to spinophilin, NF-M, V5-tag, HA-tag (as above or rabbit HA (SC-805, Santa Cruz Biotechnology)), PP1γ1, pan PP1 (SC-7482, Santa Cruz Biotechnology), PKA substrate antibody (#9624, Cell Signaling), Myc (sc-40, Santa Cruz Biotechnology), or tyrosine hydroxylase (TH) antibody (#22941, ImmunoStar, Hudson, WI). Appropriate infrared secondary antibodies were used (donkey anti-goat, donkey anti-rabbit, or donkey anti-mouse conjugated to Alexa Fluor 690 or 780; ThermoFisher or Jackson Immunologicals), and fluorescence intensity measurements were made using Image Studio (LI-COR Biosciences, Lincoln, NE).

All the formalin-fixed paraffin-embedded (FFPE) slides were prepared and stained by the Pathology Core Facility at COH using a standard protocol. Antibodies used in this study were: rabbit polyclonal antibodyWNT5B (SAB2900204) (1:20 dilution, Sigma-Aldrich), mouse monoclonal antibody Myc (SC-40) (1:75 dilution, Santa Cruz Biotechnology) and rabbit monoclonal antibody MCL1 (ab32087) (1: 100 dilution, Abcam). All antibodies were titrated with negative and positive controls to obtain optimal staining.

Cell lysates were prepared and subjected to western analysis as described previously (Williams et al., 1993 (link)) using antibodies to the following: α-tubulin (T9026; Sigma-Aldrich; 1:10,000), ASCL2 (4418; EMD Millipore, Watford, UK; 1:500), β-catenin (9587; Cell Signaling Technology, MA, USA; 1:5000), β-catenin (610153; BD Biosciences, CA, USA; 1:10,000), de-phosphorylated (actively signalling) β-catenin (05-665; EMD Millipore; 1:1000), BCL-3 (ab49470; Abcam, Cambridge, UK; 1:1000), BCL-3 (23959; Proteintech, Manchester, UK; 1:2000), MYC (sc-40; Santa Cruz Biotechnology, TX, USA; 1:500), cyclin D1 (2978; Cell Signaling Technology; 1:1000), lamin A/C (4200236; Sigma; 1:5000), LEF1 (2230; Cell Signaling Technology; 1:1000), LGR5 (ab75850; Abcam; 1:1000), NF-κB1 (p50; sc-8414; Santa Cruz Biotechnology; 1:1000), NF-κB2 (p52;05-361; EMD Millipore; 1:1000) and TCF4 (2569; Cell Signalling Technology; 1:1000).