Supplementary Figure 6: RNF2 promotes dephosphorylation of STAT1

(a) Immunoprecipitation analysis of the association between STAT1 and JAK1 in L929 cells transfected with Flag-STAT1, with or without HA-RNF2, stimulated with mouse IFN-β (1 ng/ml) for 1 h. (b) Immunoprecipitation analysis of the association of STAT1-STAT2 or STAT1-RNF2 in L929 cells co-transfected with HA-Ub-K33O or not, with or without V5-RNF2, stimulated with mouse IFN-β (1 ng/ml) for 3 h. (c) Immunoblot analysis of pY-STAT1 and pY-STAT2 in RAW264.7 and RNF2-overexpressing cells stimulated with mouse IFN-β (500 pg/ml) for indicated times. (d) Immunoblot analysis of pY-STAT1 in macrophages from Rnf2 ^fl/fl and Rnf2 ^fl/fl Lyz2-cre mice stimulated with mouse IFN-γ (500 pg/ml) for indicated times. (e) Immunoblot analysis of protein level of STAT1 and RNF2 in si-RNA-transfected macrophages from Ifnar1 ^-/- mice treated with cycloheximide (CHX) (100 μg/ml) for indicated hours 3 h post IFN-β stimulation (500 pg/ml). (f) Immunoblot analysis of pY-STAT1 and pY-STAT2 at indicated times upon addition of staurosporine (100 nM) in RAW264.7 and RNF2-overexpressing cells 30 min post mouse IFN-β stimulation (500 pg/ml). The image intensity of pY-STAT1 and pY-STAT2 was quantified in below. Data are representative of three independent experiments (a-f), pooled from three independent experiments (f) (shown as mean and SEM in f), * P < 0.05, ** P < 0.01, two-tailed unpaired Student’s t-test