Typhoon trio variable mode imager

Cells were starved in methionine and cysteine free RPMI for 30 min at 37°C, then labelled using EasyTag Express [35S]-protein labeling mix (Perkin Elmer) for 60 min at 37°C. Cells were lysed in 1% Triton X-100 (Sigma) TBS containing 10 mM NEM, 1 mM PMSF and protease inhibitors for 30 min at 4°C. Equal aliquots of clarified cell lysates were either kept at 4°C or heated at 22, 37 or 50°C for 12 min, before returning to 4°C. Immunoprecipitation and SDS-PAGE were performed as above. Gels were subsequently fixed in 12% acetic acid, 40% methanol and dried. Images were obtained using a phosphor screen (Perkin-Elmer) or on film. PhosphorImager analysis was performed using Typhoon Trio variable mode imager (GE Healthcare) together with ImageQuantTL software. Densitometry of the MHC class I HC band was performed. The amount of recoverable HLA-A2 remaining at each temperature was determined as a percentage of the signal intensity at 4°C. Graphs were generated using GraphPad Prism  software. High-resolution images were obtained using film.

Electrophoretic mobility shift assays were used to monitor the interactions between STING proteins and cyclic dinucleotides as previously described (Morehouse et al., 2020 (link)). Briefly, 20 nM of each α-³²P labeled cyclic dinucleotide was incubated with STING proteins at indicated concentrations or with serial dilutions of STING protein ranging from 0.5 nM to 50 μM in a buffer containing 5 mM magnesium acetate, 50 mM Tris-HCl pH 7.5, 50 mM KCl, and 1 mM TCEP. Reactions were incubated at room temperature for 20 min before resolving on a 7.2-cm 6% nondenaturing polyacrylamide gel run at 100 V for 45 min in 0.5× TBE buffer. The gel was fixed for 15 min in a solution of 40% ethanol and 10% acetic acid before drying at 80°C for 45 min. The dried gel was exposed to a phosphor-screen and imaged on a Typhoon Trio Variable Mode Imager (GE Healthcare). Signal intensity was quantified using ImageQuant 5.2 software.

Reaction mixtures containing 0.5 nM Cy5-labeled NonEQ DF-6,1_dsDNA (Cy5 was placed 15-nt away from the nick junction on the downstream dsDNA on the 5’flap primer) in 1X reaction buffer (50 mM HEPES-KOH pH 7.5, 100 mM KCl, 0.1 mg/ml BSA, 5%(v/v) glycerol, 10 mM MgCl₂ and 1 mM DTT) were pre-incubated at 37°C before the initiation of the cleavage reaction with the addition of varying concentrations of FEN1. Each reaction mixture was incubated further at 37°C and equal aliquots were removed and quenched by equal volumes of 2X denaturing buffer (90% deionized formamide, 100 mM EDTA) at the following time intervals (0, 0.17, 0.5, 1, 1.5, 2, 5, 10 mins). These samples were run on 20% denaturing PAGE gels, which were imaged using a Typhoon TRIO Variable Mode Imager (GE Healthcare, Life Sciences). The product formation was quantified using the ImageJ gel analysis tool. For each FEN1 concentration, the concentration of the product formed was plotted against time to estimate the initial rate (v₀, nM.min⁻¹) by taking the slope of the linear part. These v₀ values were plotted against the FEN1 concentration and K_m was determined by nonlinear least-squares fitting using a Michaelis-Menten model.

Binding assays were performed with 28 bp specific/non-specific DNA in binding buffer (50 mM Tris pH 8.0, 10 mM MgCl₂, 1 mM DTT, 150 mM KCl). 250 nM DNA was allowed to bind with varying concentrations of LlaBIII or LlaBIII^ΔLoop (0, 10, 50, 100, 250, 500, 1000, 2500, 5000 nM). Protein and DNA were mixed and incubated on ice for 10 min. Reactions were stopped by adding half the volume of STB (0.1 M Tris pH 7.5, 40% w/v sucrose, 0.4 mg/ml bromophenol blue) and loaded on 6% native PAGE run at 4°C. The gel was stained with ethidium bromide and imaged on Typhoon TRIO+ variable mode imager (GE Healthcare).

EMSA was done as described previously (Rio, 2014 (link), Zheng et al., 2012 (link)), with minor modifications. Briefly, 20 μL mixtures containing 20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 2.2 mM MgCl₂, 0.5 mM DTT, 0.2 mM EDTA, 20% (w/v) glycerol, and 50 nM [α-³²P]-labeled PNCTR fragment were incubated for 30 min at 30°C with 0-1 μM purified recombinant PTBP1 or equal amounts of bovine serum albumin (BSA; Sigma-Aldrich; cat# 10711454001). For competition assays, 0-4 μM of unlabeled RNA probes were pre-incubated with 75 nM of PTBP1 protein for 30 min at 30°C before adding 50 nM of [α-³²P]-PNCTR fragment probe and continuing the incubation for another 30 min. The RNA-protein complexes were separated by electrophoresis in 6% native polyacrylamide gels and the visualized using a Typhoon Trio Variable Mode Imager (GE Healthcare Life Sciences).