Figure 2

IRF5 localizes in the cytoplasm and lacks tumor-suppressor activity in thyroid cancer cells. A. Cytoplasmic (C), soluble (NX1) and insoluble (NX2) nuclear fractions were isolated from the indicated cell lines before and after a 24 hour treatment with IFNα. The different lysates were then blotted for IRF5. Tubulin (Tub) and histone 2B (H2B) (both from Santa Cruz) confirmed the purity of cytoplasmic and nuclear extracts. B. The same cells were subjected to IF for IRF-5 using the indicated secondary antibody. Hoechst and phalloidin (Alexa Fluor 488) were employed to identify the nuclear and cytoplasmic compartments, respectively. C. Thyroid cancer cell lines were also co-transfected with a reporter construct for p21 and p53-GFP or IRF5-GFP. After 48 hours, lysates were assayed for their relative luciferase activity expressed as fold activation over control cells (p21Luc + EV, arbitrarily set at 1). Results shown represent the average of three independent experiments. D. Alternatively, the same co-transfected cells were blotted using either anti-p21 (Santa Cruz) or anti-GFP antibodies (Covance), the latter to visualize either p53 or IRF5.