Dynabeads protein g

After transfection for 36–48 h, HEK293 cells were lysed in 700 μL lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM PMSF, with complete mini EDTA-free protease inhibitor cocktail tablets(Roche)) for 15 min and then scraped into 1.5 mL tube, incubated for 30 min with gentle agitation at 4°C. Insoluble material was removed by centrifugation (14,000 rpm) at 4°C for 20 minutes. Supernatants were collected and pre-cleared with dynabeads protein G (Novex by Life Technologies), 40 μL sample was used for input and the rest for Co-IP. Extracted proteins were incubated with 2 μg of mouse monoclonal anti-HA tag (Millipore) at 4°C overnight with gentle agitation on a platform shaker, followed by incubation with dynabeads protein G for 3 h. The beads were washed with lysis buffer 3 times to remove nonspecific binding. Immunoprecipitates were prepared in 40 μL of 2 x SDS-PAGE loading buffer (2% SDS (w/v), 20% glycerol (v/v), 2% β-mercaptoethanol (v/v), 0.2% bromphenol blue (w/v), and 100 mM Tris-HCl (pH 6.8)) for 30 min at room temperature and resolved on a 10% or 15% SDS-PAGE gel. Immunoprecipitations with mouse normal IgG (IgG-IP) were used as negative controls.

Radiolabeled PC3 or K562C cells were lysed in buffer with detergent (25 mM HEPES pH 7.6, 100 mM NaCl, 0.5% Nonidet P-40, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, protease inhibitor cocktail). Lysates were incubated with rotation at 4°C for 10 min and nuclei and other organelles were removed by centrifugation at 13 000 g for 15 min at 4 °C. Equal amounts of protein (1–2 mg) were precleared by incubation with 15–30 μl of magnetic beads (Dynabeads Protein G -Millipore) for 45 min at 4°C. Subsequently, the supernatant was incubated with 8 μg of anti-eEF1A (Millipore) or 4 μg of anti-SOD1 (Millipore) overnight at 4°C under agitation. The extracts were then incubated for 60 min with 50 μl of Dynabeads Protein G. The immunocomplexes were isolated by a magnetic support and washed five times with 500 μl of lysis buffer. Proteins bound to the beads were eluted in SDS-PAGE sample buffer and heated at 90°C for 10 min. Immunoprecipitated proteins were separated on 4–12% NuPAGE Bis-Tris gel (Invitrogen) and electroblotted onto nitrocellulose (Protran; Schleicher and Schuell). Newly synthesized proteins were detected by exposing the membrane to a Phosphorimager screen at room temperature for 16 h; while total protein was detected by incubating with antibody against EF1A or SOD. Band intensities were quantified by densitometry using with the ImageQuant software (GE Healthcare).

HEK-293T cells were stably infected with HA-I329L recombinant lentivirus (pHR-CMV-I329LeGFP), and as a negative control, with the control “empty” recombinant lentivirus (pHR-CMV-eGFP), and then transfected with TLR3-Flag expression plasmid, according to the Fugene 6 (Promega, Madison, WI, USA) protocol. At 48 h post-transfection, the cells were harvested and lysed in 1% digitonin lysis buffer (1% digitonin, 50 mM Iodoacetamide and 20 mM Tris-HCl (pH 8.0)) containing a protease inhibitor cocktail (SIGMA). Immunoprecipitations were performed with Dynabeads protein G (Millipore, Burlington, MA, USA) and 0.5% digitonin washing buffer (0.5% digitonin and 35 mM Tris-HCl (pH 8.0)) containing a protease inhibitor cocktail (SIGMA), using mouse anti-human TLR3 (Thermofisher, Waltham, MA, USA) and mouse anti-HA (SIGMA). Immunoprecipitates were resolved on a 10% SDS-PAGE gel and Western blot was performed using: mouse anti-human TLR3 (Thermofisher) followed by horseradish peroxidase goat anti-mouse secondary antibodies (Invitrogen), or rat anti-HA-HRP conjugated (Roche) to detect I329L.