Phosphatase inhibitor mini tablet

DMSO, BSA, human insulin solution, sodium selenite, kanamycin and streptomycin were from Sigma-Aldrich. l-Glutathione oxidized (GSSG, glutathione disulfide), l-Glutathione reduced (GSH) and NADPH were from AppliChem. DTT was from VWR. EDTA was from Thomasker. l-Cystine disodium salt and IPTG were from Thermo Fisher Scientific. l-glutamine and antibiotic PEN-STREP (penicillin and streptomycin) were from LONZA. Recombinant human HSP27 protein (ab4870) was purchased from Abcam. Protein concentrations were determined using the Pierce BCA assay kit. Protease inhibitor cocktail and phosphatase inhibitor mini tablets were from Thermo Fisher Scientific.

Sciatic nerves were homogenized at 4°C in RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate SDS, and 5 mM EGTA) containing protease inhibitors (Mini Protease Inhibitor Cocktail; Sigma-Aldrich) and phosphatase inhibitors (Phosphatase Inhibitor Mini Tablets; Fisher Scientific). We homogenized the tissue using Bullet Blender Homogenizer BBX24-CE (Next Advance) and then sonicated for 4 min (30 s on/off) using a Biorruptor Pico (Diagenode). Protein concentrations were determined by the BCA method (Thermo Scientific). 10 μg of total protein was subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted on to Protran nitrocellulose membrane (Amersham Biosciences). Membranes were blocked using 5% milk (Sigma-Aldrich) in TBS 1% and incubated for 16 hr at 4°C with the indicated primary antibody, washed and incubated with secondary antibodies, and developed with ECL Prime (Amersham). Antibodies used can be found online (Key Resources Table). We used an Amersham Imager 680 machine (Amersham) for visualization. Measurements from the proteins of interest were normalized to loading control GAPDH and/or CALNEXIN. When normalized to both loading controls, a mean between the normalization with GAPDH and the normalization with CALNEXIN was used for analysis. The whole membrane Western blot images are shown in source data file four online.

Sucrose gradient-purified intracellular virions of IHD-J and IHD-J-ISG15 strains, previously sonicated, were resuspended in a 50 mM Tris-HCl (pH 7.5) buffer, containing 150 mM NaCl and 1% NP-40 detergent and supplemented with a protease inhibitor cocktail mini-tablet (complete from Roche) and a phosphatase inhibitor mini-tablet (Pierce from Thermo Scientific) according to the manufacturer’s instructions. The virus extract was vortexed and incubated on ice for 15 min. The amount of protein in the samples was determined by the Bradford protein method. For immunoprecipitation, 70 μg of protein extract was incubated with the anti-V5 antibody (diluted 1:50) overnight at 4°C; then, protein G-Sepharose beads were added to the mixture, and the sample was further incubated for 2 h at 4°C. The beads were washed four times with a 50 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl and 0.1% NP-40 detergent and finally washed once with PBS. The immunoprecipitated proteins were eluted with SDS sample buffer and analyzed by immunoblotting.

Using gentle MACS dissociater,75 mg of in vivo–treated 30200 and HGS2 tumors were homogenized and lysed in 1 mL of cold RIPA lysis buffer and extraction buffer (Thermo Fisher Scientific) containing Pierce Protease and Phosphatase Inhibitor Mini Tablet (Thermo Fisher Scientific). The lysates were then centrifuged at 1,500 rpm for 10 minutes and supernatants were collected. Samples were always kept on ice between each step. Then, using a probe sonicator set at 40% amplitude, the supernatants were sonicated for 10 to 20 seconds bursts. Sonicated samples were then left on a roller for 30 minutes at 4°C. The samples were then centrifuged for 15 minutes at 13,500 rpm at 4°C. Pellets were then discarded and the samples were stored at −20°C.