Journal: The EMBO Journal
Article Title: RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity
Figure Lengend Snippet: TBK1 phosphorylates RKIP at serine 109 BMDM cells were infected with VSV (MOI 0.1) or HSV (MOI 5) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies. BMDM cells were transfected with control or TBK1 siRNA. 72 h after transfection, cells were analyzed by immunoblotting with antibodies for phospho‐RKIP or TBK1. Immunoblot analysis of RKIP phosphorylation in control or TBK1 −/− mouse embryonic fibroblasts after VSV (MOI 0.1) infection for the indicated times. Recombinant flag‐tagged TBK1 was immunoprecipitated with anti‐flag M2 beads in flag‐TBK1‐transfected 293T cells. Followed this, in vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST‐RKIP, or 100 μM GST‐GFP together with 5 μM flag‐TBK1 at 30°C for 30 min. Assay mixtures were immunoblotted with the indicated antibodies for phospho‐RKIP, flag, GST, or his. Immunoblot analysis of extracts of RAW264.7 cells stably overexpressing flag‐RKIP, flag‐S109A, and flag‐S109D, and infected with VSV (MOI 0.1) for the indicated times, followed by immunoprecipitation with anti‐flag beads. BMDM cells were infected with VSV (MOI 0.1) for the indicated times. The cell lysates were immunoprecipitated with anti‐TBK1 (left) or anti‐RKIP (right). The immunoprecipitates were analyzed by immunoblot with anti‐RKIP or anti‐TBK1 antibody. The levels of the endogenous RKIP, TBK1, and p‐RKIP were detected by immunoblot analysis. Co‐immunoprecipitation and immunoblot analysis of 293T cells transfected with myc‐TBK1 alone or with flag‐RKIP, flag‐S109A, or flag‐S109D. GST pull‐down experiment was performed with 1 μg fusion protein GST‐GFP, GST‐his‐RKIP, or GST‐his‐S109D mixed with 20 μl pre‐cleared agarose beads in 800 μl reaction medium, followed by adding 1 μg flag‐TBK1 and incubating at 4°C for 3 h with gentle rotation. Pull‐down (lane 1–3) and input samples (lane 4) were separated by SDS–PAGE followed by immunoblotting with anti‐GST or anti‐flag antibody. Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc‐TBK1, flag‐RKIP, flag‐S109A, flag‐S109D, HA‐GSK3β or flag‐GFP. In vitro assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST‐TBK1 together with 100 μM GST‐his‐RKIP, 100 μM GST‐his‐S109D, 100 μM GST‐his‐S109A, or 100 μM GST, respectively, at 30°C for 30 min, followed by immunoblotting with anti‐p‐TBK1 or anti‐GST antibody. . Source data are available online for this figure.
Article Snippet: The fusion proteins of GST‐GFP, GST‐his‐RKIP, and GST‐his‐S109D were expressed in E. coli and purified according to standard protocols with pre‐equilibrated glutathione–Sepharose beads (GE Healthcare).
Techniques: Infection, Transfection, Recombinant, Immunoprecipitation, In Vitro, Kinase Assay, Stable Transfection, SDS Page, Plasmid Preparation