Phosphorimager plate

Radioimmunoprecipitation analysis was used to examine Gag proteolytic processing and virus release efficiency. Compounds were used throughout the experiment at 5 μM or the indicated concentrations and were prepared immediately prior to use and mixed by vortexing. Metabolic labeling of HeLa cells, preparation of cell and virus lysates, and immunoprecipitation methods were described in detail previously (67 (link)). In brief, HeLa cells were transfected with the pNL4-3 WT or a mutant derivative. Transfected HeLa cells were starved in Cys-Met-free medium for 30 min and then metabolically radiolabeled for 2 h with [³⁵S]Cys-Met Promix (PerkinElmer). Ultracentrifugation was used to pellet virions. Cell and virus lysates were immunoprecipitated with pooled immunoglobulin from HIV-1-infected patients (HIV-Ig) obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID. The radioimmunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and exposed to X-ray film and a phosphorimager plate (Fuji), and the bands were quantified by using a Fujifilm FLA-5000 phosphorimager and ImageStudio software (Li-Cor).

A concentration of 0–2 mM substrate peptide (derived from STAT5b) was incubated with 5–10 nM JAK1^KD_, JAK2^KD_, JAK3^KD or TYK2^KD with varying concentrations of SOCS proteins at 25 °C for 10–30 min in 20 mM Tris pH 7.5, 150 mM NaCl, 2 mM β-mercaptoethanol, 0.1 mg/mL BSA, 2 mM MgCl₂ 1 mM ATP supplemented with 1 μCi ³²P-γ-ATP. Following this, the reactions were spotted onto P81 phosphocellulose ion-exchange paper and quenched in 5% H₃PO₄. The paper was washed (4 × 200 ml, 15 mins) with 5% H₃PO_4, dried and then and exposed to a phosphorimager plate (Fuji).