Figure 6.

hCAF1 physically interacts with STAT1. (A) Direct interaction between hCAF1 and STAT1 was analysed by GST-pulldown experiments. In vitro translated STAT1 was incubated with equivalent amounts of GST, GST-CAF1 and GST-CCR4 (Supplementary Figure 5A) bounded to glutathione-Sepharose beads. The eluted proteins were analysed by immunoblotting using anti-STAT1 antibody. (B) Endogenous hCAF1 and STAT1 interact. Extracts from MCF7 cells treated with STAT1 and hCAF1-specific siRNAs or control (siControl) siRNA were immunoprecipitated (IP) with either normal rabbit IgG or anti-hCAF1 antibody. Immunoprecipitates were then analysed by immunoblotting with anti-STAT1 antibody. (C) MCF7 protein extracts were treated with or without RNAseA before immunoprecipitation (IP) with an anti-hCAF1 antibody followed by western blot with anti STAT1 antibody. (D) STAT1 and hCAF1 co-localize in the cytoplasm of the unstimulated MCF7 cells. MCF7 were grown on coverslips in 12-well plates and then treated with 5 ng/ml of Interferon γ for 1 h. Proximity Ligation Assay (PLA) was used to detect the cellular co-localization of endogenous hCAF1 and STAT1 according to manufacturer’s instructions (Duolink, Eurogentech). (a) PLA using anti-hCAF1 and anti-STAT1 on untreated MCF7 cells and (b) MCF7 cells treated with 5 ng/ml of Interferon γ for 1 h. (E) PLA control on MCF7 cells transfected with (a) control siRNA or with (b) siRNA against STAT1. Data are representative of at least three independent experiments. Scale bar=20 μm.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure 6A ^{(48.7KB, pdf)}

Source Data for Figure 6B ^{(563.3KB, pdf)}

Source Data for Figure 6C ^{(567.4KB, pdf)}