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M35 does not target the phosphorylation or nuclear translocation of key transcription factors.

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posted on 2017-05-25, 17:59 authored by Baca Chan, Vladimir Gonçalves Magalhães, Niels A. W. Lemmermann, Vanda Juranić Lisnić, Markus Stempel, Kendra A. Bussey, Elisa Reimer, Jürgen Podlech, Stefan Lienenklaus, Matthias J. Reddehase, Stipan Jonjić, Melanie M. Brinkmann

(A) Immortalized BMDM stably expressing LacZ-myc or M35-myc were stimulated by addition of 3 μg/ml cGAMP and cells lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous IRF3 and phospho-IRF3 were detected with specific antibodies. Expression of myc-tagged LacZ and M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (B) NIH3T3 fibroblasts stably expressing myc-tagged LacZ or M35 and eGFP-IRF3 were stimulated by transfection of 10 μg/ml poly(I:C) with Lipofectamine 2000. At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear IRF3 (lower panel) are represented as the percentage of total cells (with a minimum of 100 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments. (C) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated by transfection of 10 μg/ml poly(I:C) in the presence of Lipofectamine 2000 and cells were lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous p65, phospho-p65 and tubulin were detected with specific antibodies. Expression of myc-tagged M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (D) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated as for (B). At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and a rabbit anti-p65 antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear p65 (lower panel) are represented as percentage of total cells (with a minimum of 300 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments.

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