Luminol enhancer solution

Cells were disrupted on ice in 1% NP40 lysis buffer or NETN buffer (Bio-Rad) as previously described.^{28 (link)} Protein concentration was determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted with specific primary and secondary antibodies, and detected by chemiluminescence with the ECL detection reagents, Immobilon Western HRP substrate Luminol Reagent (Millipore) and Luminol Enhancer Solution (Thermo Fisher Scientific). The membranes were imaged on the ChemiDoc Touch Imaging System (Bio-Rad). Representative blots of 3–4 independent experiments are shown. Relative changes in p-RPS6 were quantitated by densitometry analysis using Image J as a function of concentration or treatment time and averaged across all blots.

Equal amounts of protein (20 μg) were separated by SDS-PAGE (Pierce, Rockford, IL) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Blots were blocked for 1 h at room temperature in TBS-T (150 mM NaCl, 10 mM Tris, pH 8.0, 0.1% Tween 20) containing 5% nonfat dry milk. Blots were incubated with polyclonal antibodies (sc-109, Santa Cruz, CA) in TBS-T containing 5% nonfat dry milk overnight at 4°C. The blots were incubated with a horseradish peroxidase-conjugated (HRP) goat anti-rabbit antibody (Rockland, Gilbertsville, PA) for 1 h at room temperature after several washes with TBS-T. Then blots were washed in TBS-T and proteins were visualized by exposure to Luminol Enhancer Solution (Thermo Scientific Rockford, IL) according to the manufacturer’s instructions. The blots were stripped by incubation with Restore Western Blot stripping Buffer (Thermo Scientific Rockford, IL) at room temperature for 10 min. β-Actin (Santa Cruz, CA) was used to verify equal protein loading. Quantification of band intensity was obtained by using a calibrated densitometer with Quantity One software (GS800, Bio-Rad, Hercules, CA). Immunoblot results are expressed as relative densitometry units (RDU) normalized to β-Actin.

Nuclei extracted from intact 293T cells as described above were lysed by vortexing for 10 s with 1 ml of lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl) containing 0.5% IGEPAL CA-630 (Sigma), 1 mM PMSF and 20 U SUPERasin (Ambion), incubated for 10 min on ice and centrifuged at 12 000 × g for 10 min at 4°C. For immunoprecipitation of endogenous protein complexes, the prepared nuclear lysate (1.5 mg) was incubated with 3 μg of rabbit normal IgG (Active Motif) or rabbit anti-RRP1 (GeneTex, 115107) for 4 h at 4°C. The antibody-bound RRP1-associated protein complexes were incubated with 10 μl of Dynabeads Protein G (Invitrogen) for 1 h at 4°C, and then the complex-associated beads were washed five times with lysis buffer containing 0.5% IGEPAL CA-630, once with lysis buffer, and were eluted using 2% (w/v) SDS sample buffer. Immunoblotting was performed as described (20 ,21 (link)) using an EasyBlot kit (GeneTex). Signal was detected using Luminol/Enhancer solution and Stable Peroxide solution (Thermo Scientific) as a substrate for peroxidase and using an LAS4000 image analyzer.

Cells were lysed in RIPA lysis buffer (20 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1% NP-40 or Triton -100; 0.5% sodium deoxycholate and 0.1% SDS) with 1 mM PMSF and a protease inhibitor (Sigma cat. No. S8830). Prior to separation, × 2 Laemmli sample buffer ]0.125 M Tris HCl, pH 6.8; 20% glycerol; 4% SDS; 0.004% bromophenol blue and 5% 2-mercaptoethanol (BME)] was added in a 1: 1 ratio. Subsequently, the samples were incubated for 5 min at 95 °C. 40 µg/lane of cell lysates was loaded on a 12% gel SDS-PAGE. The proteins were transferred to 0.2 m nitrocellulose membrane for 1 h at 100 V. The membrane was blocked using blocking buffer solution [(5% BSA in TBST (Tris-buffered saline, 0.1% Tween)] for 1 h at room temperature. The primary antibody, rabbit anti-LC3B (NB100-2220 Novus biologicals, USA, ~ 2 g/mL) was diluted in blocking buffer and incubated with the membrane overnight at 4 °C. The membrane was rinsed with TBST, 3 times for 5 min each. The blot was incubated with Working Solution [(mixing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution (Thermo Scientific™, 34080)] for 5 min. Signal intensities of LC3B bands were normalized against the internal control PonceauS [(Sigma P-3504) 0.1% PonceauS and 5% Acetic acid)], which label all proteins according to their concentrations. LC3B-II runs at 14–16 kDa while LC3B-I runs at 16–18 kDa.