Pharosfx imager

The glutathione S‐transferase (GST) pull‐down assay was performed according to the previously described methods (Mehto et al, 2019a (link), 2019b ). Briefly, GST or GST‐RIPK2 or GST‐IRGM recombinant proteins were expressed in E. coli SoluBL21 (Amsbio), and the proteins were purified on Glutathione Sepharose 4 Fast‐Flow beads (GE Healthcare). The [³⁵S] labeled‐ Myc‐NOD1, Myc‐NOD2, Myc‐RIPK2, Myc‐ULK1, Myc‐ATG16L1 or Myc‐Beclin‐1 proteins were in vitro translated using TnT T7–coupled reticulocyte lysate system (Promega). The GST proteins were incubated with [³⁵S]‐labeled proteins in 200 μl of NETN‐E buffer (50 mmol/l Tris, pH 8.0, 100 mm NaCl, 6 mm EDTA, 0.5% NP‐40, and 1 mm dithiothreitol (DTT) supplemented with complete mini EDTA‐free protease inhibitor cocktail; Roche) for 2 h at 4°C. After incubation, the beads were washed five times with NETN‐E buffer, boiled with loading buffer, and subjected to SDS–PAGE. The gels were stained with coomassie blue and vacuum‐dried. The GST was detected by staining with coomassie blue stain, whereas the [³⁵S]‐ labeled Myc‐tagged proteins were detected in PharosFX imager (Bio‐Rad Laboratories).

GST pulldown assay was done as described previously (Mehto et al, 2019). GST‐IRGM recombinant protein was expressed in SoluBL21 (Amsbio) and purified on Glutathione Sepharose 4 Fast‐Flow beads (GE Healthcare). [³⁵S]‐labeled Myc‐cGAS, Myc‐RIG‐I, and Myc‐TLR3 were cotranscribed/translated using the TnT T7‐coupled reticulocyte lysate system (Promega). The in vitro‐translated [³⁵S]‐labeled proteins were then incubated with GST or GST‐IRGM in 250 μl of NETN‐E buffer (50 mM Tris pH 8.0, 100 mM NaCl, 6 mM EDTA, 6 mM EGTA, 0.5% NP‐40, and 1 mM dithiothreitol supplemented with Complete mini EDTA‐free protease inhibitor cocktail [Roche]) for 2 h at 4°C and then washed five times with 1 ml of NETN‐E buffer, boiled with loading buffer, and subjected to SDS–PAGE. The gel was stained with Coomassie Blue and vacuum‐dried. The GST‐IRGM was detected by staining with Coomassie Blue, whereas the [³⁵S]‐labeled PRRs were detected in PharosFX imager (Bio‐Rad Laboratories).

293 T cells not expressing or expressing SAMHD1-GFP WT, K354R, K354Q, or HD/AA were washed in cold PBS and then resuspended in 65% methanol for lysis. Cell lysate was heated to 95 °C, clarified by centrifugation, dried in speed vac and resuspended in water. Quantification of cellular dNTP pool was carried out as outlined previously^{31 (link)}. Briefly, ³²P-5′ labeled primer (18 nucleotide) was annealed to four different 19-nucleotide templates creating nucleotide variation at the template 5′ end. dNTPs from cell extracts were used to extend a single nucleotide at the 5′ end of the primer. Products were ran on urea-PAGE gels and amount of extended primers for each of the 4 variations was quantified with BioRad PharosFX imager and Image Lab software (version 5.12). Amount dATP, dGTP, dTTP, and dCTP in cells were expressed as pmol/10⁶ cells.

GST pulldown assay was done as described previously (Kimura et al., 2015 (link), Chauhan et al., 2016 (link)). GST-IRGM recombinant protein was expressed in SoluBL21 (Amsbio) and purified on Glutathione Sepharose 4 Fast-Flow beads (GE Healthcare). [35S]-labeled Myc-NLRP3 was cotranscribed/translated using the TnT T7–coupled reticulocyte lysate system (Promega). The in vitro–translated [35S]-labeled Myc-NLRP3 protein was then incubated with GST or GST-IRGM in 250 μl of NETN-E buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 6 mm EDTA, 6 mm EGTA, 0.5% NP-40, and 1 mm dithiothreitol supplemented with complete mini EDTA-free protease inhibitor cocktail [Roche]) for 2 h at 4°C and then washed five times with 1 mL of NETN-E buffer, boiled with loading buffer, and subjected to SDS-PAGE. Gel was stained with Coomassie Blue and vacuum-dried. The GST-IRGM was detected by staining with Coomassie Blue, whereas the [35S]-labeled Myc-NLRP3 was detected in PharosFX imager (Bio-Rad Laboratories)

Helicase assay reaction mixtures (20 μL) contained 40 mM Tris HCl (pH 7.4), 25 mM KCl, 5 mM MgCl₂, 2 mM dithiothreitol, 2% glycerol, 100 ng/uL BSA, 2 mM ATP, 0.5 nM of DNA substrate (forked duplex or G4 substrate), and the indicated concentrations of ChlR1. Helicase reactions were initiated by the addition of ChlR1, incubated at 37°C for 20 min, and terminated by addition of Stop buffer containing EDTA. Reaction products were resolved on nondenaturing 12% (19:1 acrylamide/bisacrylamide) polyacrylamide gels for the forked duplex substrates and nondenaturing 8% (19:1 acrylamide/bisacrylamide) polyacrylamide gels, as described previously [32 (link)]. For triplex DNA, helicase assay reaction mixtures (20 μL) contained 25 mM HEPES (pH 7.5), 25 mM potassium acetate, 1 mM magnesium acetate, 1 mM DTT, 100 μg/mL BSA, 1 mM ATP, 0.5 nM of the specified triplex DNA, and the indicated concentrations of ChlR1 protein. The products of the helicase reactions were resolved on nondenaturing 10% (19:1 acrylamide:bisacrylamide) polyacrylamide gels with 40 mM Tris acetate (pH 5.5) and 25% glycerol and running buffer containing 40 mM Tris acetate, pH 5.5, and 5 mM MgCl₂. Radiolabeled DNA species in polyacrylamide gels were visualized using a PharosFX Imager and quantified using Quantity One software (Bio-Rad).