Ecl detection system

Western blots were performed as previously described elsewhere. Membranes were incubated with the following primary antibodies: anti‐MYBBP1A (Proteintech #14524‐AP, Rosemont, IL, USA ), anti‐PGC1α (Abcam #ab54481, Cambridge, UK), anti‐SGLT1 (Abcam #ab14685), anti‐p38 MAPK (Cell Signaling #9212, Danvers, MA, USA), and anti‐phospho‐p38 MAPK (T180/Y182) (Cell Signaling #9215). α‐Tubulin (Sigma #T9026) was used as a loading control. Horseradish peroxidase‐labeled rabbit anti‐mouse (Abcam #ab 97046) and goat anti‐rabbit (Abcam #ab 97051) secondary antibodies were used. The proteins were detected using an ECL detection system (Amersham Biosciences) and Bio‐Rad ChemiDoc XRST (Berkeley, CA, USA).

The membrane fraction samples (input samples) and the samples obtained after CoIP were subjected to SDS-PAGE using a Tris-glycine gel. The samples were mixed with the loading buffer and 2-mercaptoethanol and heated at 95 °C for 5 min prior to loading. After electrophoretic separation, proteins were electrophoretically transferred onto PVDF membrane. The membrane was blocked with 5% (w/v) skim milk in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl containing 0.1% (v/v) Tween-20 (TBS-T buffer) for 60 min. After washing the membrane with TBS-T, primary antibodies, diluted in TBS-T containing 5% (w/v) skim milk, were added, and the membrane was incubated overnight at 4 °C. Anti-DDDDK-tag mAb HRP-DirecT (MBL), which binds to the FLAG tag, was used to detect PRELP, IGF-I Receptor β rabbit mAb (Cell Signaling Technology) was used to detect IGFI-R, and p75NTR rabbit mAb (Cell Signaling Technology) was used to detect p75NTR. As a loading control for input samples, Na,K-ATPase was detected by Na,K-ATPase α1 rabbit mAb (Cell Signaling Technology). Anti-DDDDK-tag mAb HRP-DirecT was detected by the ECL detection system (Amersham) according to the manufacturer's instruction. The primary antibodies that recognize IGFI-R, p75NTR, and Na,K-ATPase were detected using anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology) using the ECL detection system.

IP and immunobloting experiments were performed as previously described (42 (link)). O-GlcNAc was enriched by treating the cells with Glu plus TMG as previously described (treated with 5 μM TMG for 24 h and 30 mM Glu for 3 h) (41 (link)). Antibodies used were as follows: anti-ULK1 (CST; catalog no.: 8054), anti-OGT (Santa Cruz; catalog no.: sc-32921), anti-O-GlcNAc (RL2) (Abcam; catalog no.: ab2739), anti-O-GlcNAc (CTD110.6) (Sigma; catalog no.: O7764), LC3B (CST; catalog no.: 2775S), and anti-STX17 (Merck; catalog no.: HPA001204).
Peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch. Blotted proteins were visualized using the ECL detection system (Amersham). Signals were detected by a LAS-4000 (Fujifilm) and quantitatively analyzed by densitometry using the Multi Gauge software (Fujifilm). All Western blots were repeated at least three times. Silver staining analysis was performed as described (41 (link)).

The nitrocellulose membrane carrying transferred proteins was incubated at 4 °C overnight with corresponding antibody at 1:1000 ratio. Immunodetection was accomplished using horseradish peroxidase-conjugated secondary antibody, followed by ECL detection system (Amersham).

For immunoblotting, cell protein extracts were prepared following standard procedures in RIPA buffer in the presence of protease inhibitors (Complete, Roche Diagnostics) and phosphatase inhibitors (PhosStop, Roche Diagnostics). After quantitation with the Micro‐BCA Protein Assay Kit (Thermo Fisher Scientific), 25 µg of protein was separated by SDS–PAGE and transferred to PVDF membranes (Immobilon‐P, Millipore). After using appropriated primary and secondary antibodies, blots were developed by a peroxidase reaction using ECL detection system (Amersham‐G.E. Healthcare). Antibodies used were Recombinant Anti‐NeuroD1 antibody [EPR4008] (ab109224; 1:1,000 dilution), Anti‐MASH1/Achaete‐scute homolog 1 (Abcam ab211327; 1:1,000 dilution), Mouse Anti‐RNAPII 7c2 (IGBMC Antibody facility; 1:1,000 dilution), and Anti‐Phospho‐Serine 2 RNAPII (Abcam ab5095; 1:1,000 dilution). Anti α‐Tubulin (SIGMA T5168) (Novus Biologicals hVIN‐1 NB600‐1293; 1:1,000 dilution) was used as loading control. All the blots are representative of at least three independent experiments.

Protein expression was evaluated from cell lysates or concentrated supernatants (ultracentrifugation at 25,000 rpm for 1 h) by Western blot analysis. Protein concentrations were determined with the Pierce BCA Protein-Assay Kit (Thermo Fisher Scientific, Schwerte, Germany) and 20 µg protein per sample were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Primary antibodies were directed against CHIKV E2 (Eurogentec, Cologne, Germany; custom made), RRV E2 (ATCC VR-1246AF), and β-actin (Sigma, Munich, Germany; no. A5441). After incubation with appropriate secondary horseradish peroxidase (HRP)-coupled antibodies, proteins were detected with the ECL detection system (Amersham, Freiburg, Germany) and the Fusion FX7 imaging system (Vilber, Eberhardzell, Germany).

Western blot analyses were performed as previously described [50 , 51 ]. Briefly, the cells were washed twice with PBS and lysed via sonication in lysis buffer (50 mM Tris-HCl, pH 7.5; 1% NP-40; 1 mM Na3VO4; 150 mM NaCl; 20 mM Na4P2O7; 100 mM NaF; 1% Na-deoxycholate; 0.1% SDS; 1 mM EDTA; phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma)). The samples were separated on 6–15% SDS-PAGE gels, transferred to nitrocellulose membranes (Protran BA83, Whatman) and immunostained. The following primary antibodies and dilutions were used: Anti-p21 [C-19] 1:200 (Santa Cruz, #sc-397), anti-p53 [FL-393] 1:200 (Santa Cruz, #sc-6243), Anti-p16 (M-156) 1:200 (Santa Cruz, #sc-1207), anti-pRb 1:500 (BD-Pharmingen), anti-phospho-pRb (Ser807/811) 1:1000 (Cell Signaling), anti-phospho-histone H2A.X (Ser139) 1:1000 (Millipore 05–636) and monoclonal anti-α-tubulin 1:1000 (Sigma 9026). Horseradish peroxidase-labeled rabbit anti-mouse (Amersham, diluted 1:3000) and goat anti-rabbit (Abcam, #6721, diluted 1:3000) secondary antibodies were used. The proteins were visualized using an ECL detection system (Amersham Biosciences).