Typhoon imager

All experiments were performed at least three times, the fits to data were generated by the Kaleidagraph software (Synergy). Fluorescence anisotropy-binding experiments were carried out in either 40 mM HEPES pH 7.4, 160 mM KCl, 5% glycerol, 0.1% Triton X-100, 1 mM EDTA, 5 mM DTT (herein referred to as ‘buffer A') or 40 mM HEPES pH 7.4, 400 mM KCl, 5% glycerol, 0.1% Triton X-100, 1 mM EDTA and 5 mM DTT (herein referred to as ‘buffer B') at room temperature as described in Morrone et al.23 (link)
Förster resonance energy transfer experiments were carried out in either buffer A (for AIM2^FL and AIM2^Hin) or buffer C (40 mM HEPES pH 7.4, 60 mM KCl, 5% glycerol, 0.1% Triton X-100, 1 mM EDTA and 5 mM DTT) for AIM2^Hin as described in Morrone et al.23 (link)
Electrophoretic mobility shift assay experiments were carried out in buffer A. To a fixed amount of fluorescein-labelled dsVACV72 was added increasing concentrations of AIM2. The reaction was allowed to equilibrate at room temperature (at least 20 min), then applied to a 4% 116:1 acrylamide:bis-acrylamide Tris-borate-EDTA gel. The gel was run at 100 V in 1 × Tris-borate-EDTA buffer and imaged using a Typhoon imager (GE Healthcare; excitation at 488 nm, emission at 532 nm).

For this analysis, 10% Tris.HCl gels (Biorad, Hercules, CA, USA) were used for gel electrophoresis following the manufacturer’s protocol. All blue precision standard (Biorad) was used as the protein standard. HPV16 VLPs were released from microparticles by resuspension in phosphate-buffered saline (PBS). Different dilutions of the resuspended vaccine particle preparations, along with a standard curve of known amounts of HPV16 VLPs spiked into blank microsphere suspension, were run on the gel. The gel was transferred onto a nitrocellulose membrane using the i-blot system (Thermo Scientific, Waltham, MA, USA). L1 protein was detected using Camvir-1 (Abcam, Cambridge, MA, USA) followed by FITC-labeled goat anti-mouse antibody. The membrane was scanned on the Typhoon imager (GE healthcare, Pittsburgh, PA, USA) and quantitative analysis of HPV16 L1 within the microparticles was conducted using ImageJ software (open-source program).

A 50 bp double-stranded DNA molecule was generated through the synthesis of two complementary single-stranded DNA molecules (Eurofins Genomics) that were annealed together by heating to 98 °C and cooling gradually to room temperature. One strand had 32 nucleotides of complementarity with the crRNA in the purified Csy complex and was labelled at the 5′ end with [³²P]. The electrophoretic mobility shift assay (EMSA) experiment was conducted in binding buffer (10 mM HEPES, pH 7.5, 1 mM MgCl₂, 20 mM KCl, 1 mM TCEP, bromophenol blue and 6% glycerol) at 37 °C for 15 min. The Csy complex was routinely used at a concentration of 100 nM in reactions, with 1 nM labelled DNA. Wild-type AcrF1 or the indicated protein variants were used at molar excess compared with the Csy complex as indicated in the relevant figures. AcrF1 was incubated with the Csy complex for 1 h at 4 °C. After the appropriate incubation, the reactions were resolved on native 6% polyacrylamide Tris-Borate-EDTA (TBE) gels. Gels were wrapped in Saran wrap and visualized with a phosphoscreen on a Typhoon imager (GE Healthcare Life Sciences).

3 μM Ssa1 with or without 3 μM Ydj1 variants in reaction buffer (20 mM K-Hepes [pH 7.5], 150 mM KOAc, 5 mM Mg(OAc)₂, 5 mM β-ME) was mixed with 2.4 μM ATP containing γ³²P-ATP and incubated at room temperature. At indicated time points, 2 μl aliquots were removed from the reaction and quenched in 4 μl TLC solution (500 mM LiCl, 1 M Formic acid). γ³²P-ATP and γ³²P-Pi were separated by thin layer chromatography using PEI cellulose (Vendor) and quantified by phosphorimaging using a Typhoon imager (GE Healthcare). Time courses of the reaction were fit to Equation 3, ${\left[ATP\right]}_t=a\times e^{-k_{obsd}t}$ in which a is the fraction of ATP before initiation of the reaction, and k_obsd is the observed rate constant for Ssa1 ATP hydrolysis.

PIP₂-containing vesicles with the composition described above were resuspended at a concentration of 1 mg/ml and phosphorylated by incubation for 2 h with PI3K, purified as detailed previously [42 (link)]. The reaction was carried out in 20 mM Tris/HCl, pH 7.4, 50 mM NaCl, 50 mM KCl, 1 mM EGTA, 3 mM MgCl₂ and 0.1 mM ATP, before being quenched with 10 mM EDTA. The resulting PIP₃-containing vesicles were then incubated with PTEN for 3 min at 25°C and quenched with 22 mM H₂O₂ (Sigma). The quenched reaction was agitated for 10 min at 500 rev./min at 25°C, before being spotted on to a nitrocellulose membrane. After the droplet had dried, the membrane was washed four times with TBS/Tween 20 solution (TBST) (50 mM Tris/HCl, pH 7.4, 150 mM NaCl and 0.1% Tween 20) over the course of 1 h, before being blocked with TBST+2 mg/ml BSA for 1 h at 4°C. The membrane was then incubated with 0.5 μg/ml Alexa Fluor 488-labelled GRP1-PH domain in TBST+2 mg/ml BSA overnight at 4°C as described previously [58 (link)]. The membrane was washed six times in TBST and dried at 25°C before being imaged using a Typhoon imager (GE Healthcare). The intensities were quantified using ImageQuant (GE Healthcare).