Extended Data Fig. 5: SHISA9 regulates antiviral and pro-inflammatory genes expression through IKKi.
From: Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice
(a) U87 was infected without or with HSV-1 (MOI = 1) for 24 hrs, the cell lysates were then immunoprecipitated (IP) by SHISA9, and the proteins binding with SHISA9 were eluted and send for mass spectrometry analysis (MS). The IKKi peptide was detected by IP-MS. Green color represents the b- fragment ions (come from the N-terminus) while orange color represents y - fragment ions (come from the C-terminus). (b) Co-immunoprecipitation (IP) and immunoblot analysis of extracts of BV2 cells with or without HSV-1 (MOI = 1) infection for 24 hrs. WCL, whole-cell lysates. (c) List of the top 100 upregulated immune genes in the brain of Shisa9−/− mice after intracranial infection with HSV-1 (1 × 102 PFU) for 7 days. IKKi-dependent genes are labelled. (d) Real-time quantitative polymerase chain reaction (qPCR) analysis of indicated genes in wild-type (WT) and Shisa9−/− astrocytes transfected with control siRNA or Ikbke siRNA, followed by HSV-1 (MOI = 1) infection for 24 hrs. The efficiency of Ikbke siRNA was confirmed by immunoblot. (e) Immunoblotting analysis of extracts of HEK293T cells transfected with HA-IKKi and Myc-SHISA9, followed by HSV-1 (MOI = 1) infection for 24 hrs, then treated with bafilomycin A1 (Baf A1, 0.2 μM), MG132 (10 μM), 3-methyladenine (3-MA, 10 mM) or lactacystin (5 μM) for 6 hrs. NT, not treated. Similar results were obtained by three independent biological experiments. In (d) all error bars, mean values ± SEM, P-values were determined by unpaired two-tailed Student’s t test of 3 independent biological experiments.
