Supersignal ecl

N/TERT-1 grown in 2D or differentiated in 3D organotypic cultures (total cultures) were lysed with Lysis Buffer containing 50 mM Tris, HCl pH 8.0, 150 mM NaCl, 1% Triton X-100 and protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was measured according the Bradford method (BioRad, Munich, Germany). The protein solution was diluted in NuPage loading buffer (Invitrogen, Carlsbad, CA, USA) and 30 or 60 µg per line loaded on 4–12% Bis-Tris NuPage pre-casted gels (Invitrogen, Carlsbad, CA, USA). The separated proteins were transferred on nitrocellulose membrane using iBlot semi-dry transfer apparatus. Membranes were blocked with blocking buffer (5% skimmed milk, 0.1% Tween 20 in PBS) for 1 h at room temperature and incubated with primary antibody against p21 (Upstate, Temecula, CA), COX-2 (Millipore, Temecula, CA) 1∶500; Phospho-p38Tyr^180/182 (Cell Signaling, Denver, MA, USA) 1∶1000; GAPDH and β-actin 1∶5000 (Sigma-Aldrich, St. Louise, MO, USA) overnight at 4°C. The membranes were washed with 0.1% PBS-T and incubated with secondary HRP- conjugated antibody (ECL™ Anti-Mouse/Anti Rabbit IgG, GE Healthcare, Little Chalfont, UK) for 1 h at room temperature. After washing with 0.1% PBS-T membranes were incubated with SuperSignal ECL (Thermo Scientific, Rockford, IL, USA) and developed on X-ray sensitive film.

Cells were treated with melatonin, 5-Fu or both before dissolved with radioimmunoprecipitation for protein extraction and separated by SDS-PAGE as previously described.^{45 (link)} Briefly, the cells were washed twice with cold PBS, scraped off the plate, pelleted and resuspended in radioimmunoprecipitation buffer. After lysis on ice for 15 min, samples were centrifuged and the supernatant collected. To quantify protein concentration of each sample, the BCA kit was used and equal amount samples were separated on 8–15% SDS-PAGE gels before transfferation to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, USA). The membranes were then blocked with 5% non-fat milk in tris buffered saline tween (TBST) for 1 h at room temperature, incubated with the indicated primary antibody diluted in 5% bovine serum albumin in TBST at 4 °C overnight. Then the membranes were washed thrice with TBST, probed with peroxidase-linked secondary antibody for 1 h at room temperature. To visualize proteins in the membrane, enhanced chemiluminescence (SuperSignal ECL, ThermoFisher Scientific, Carlsbad, USA) was used.

HEK293 cells were transfected using Lipofectamine 2000 (Thermo Scientific). After 48 hr cells were washed, collected with PBS buffer, and pelleted. Cells were then solubilized with lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM PMSF, and protease inhibitors (Hoffman La Roche, Basel, Switzerland) containing 1% β-D-dodecyl maltoside (Sigma-Aldrich, St. Louis, MO) for 30 min at RT. The lysate was centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was incubated with the indicated primary antibody for 30 min then 20 μl of protein A/G agarose beads (Thermo Scientific) were added for incubation overnight at 4°C with rotation. The beads were washed three times in washing buffer (mM) (20 HEPES, pH 7.5, 150 NaCl, 10 KCl, 2 EDTA, 10% glycerol). Proteins were eluted with 40 μl of 2X SDS sample buffer at 37°C. Bound and eluted proteins were subsequently separated by SDS-PAGE and transferred to PVDF membrane (BioRad, Hercules, CA). After blocking with 5% skimmed milk for 1 hr, the membrane was probed with the indicated antibodies over night at 4°C. HRP-conjugated secondary antibodies were applied for 30 min at RT. Blots were detected with SuperSignal ECL (Thermo Scientific) and developed with GeneMate Blue film (BioExpress, Kaysville, UT).

α-O-GlcNAc (RL2, Abcam, ab2739, 1:1000; CTD110.6, Cell Signaling, #12938, 1:10,000), α-OGT (Cell Signaling, #5368, 1:1000), α-SCD1 (ThermoFisher, PA5-17409, 1:1000), α-SCD2 (G15, Santa Cruz, sc-14722, 1:200), α-PPARγ (D69, Cell Signaling, 2430, 1:1000), α-phospho-PPARγ (AW504, Millipore, 04-816, 1:1000), α-Acetyl CoA carboxylase 1 (Cell Signaling, 4190, 1:1000), α-Fatty acid synthase (Novus, NB400-114, 1:1000), α-Nape-pld (Abcam, ab95397, 1:1000), α-FAAH (Abcam, ab54615, 1:1000), α-FLAG (M2, Sigma, A8592, 1:10,000), α-GAPDH (FL-335, Santa Cruz, sc-25778, 1:1000), and α-alpha-Tubulin (B-5-1-2, Sigma, T5168, 1:1000) were purchased from vendors. Tissues were lysed in buffer containing 1% Nonidet P-40, 50 mM Tris-HCl, 0.1 mM EDTA, 150 mM NaCl, proteinase inhibitors and protein phosphatase inhibitors. Thirty micrograms of protein lysate were electrophoresed on SDS-PAGE gels and transferred to methanol-activated PVDF membranes. Primary antibodies were incubated at 4 °C over the night. Western blotting was visualized by peroxidase conjugated secondary antibodies and ECL chemiluminescent substrate. Uncropped western blots images were shown in Supplementary Fig. 12 and Suplementery Fig. 17. O-GlcNAc signals detected by CTD110.6 antibody were visualized by SuperSignal ECL (ThermoFisher, 34095). Experiments were repeated three times.