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. Author manuscript; available in PMC: 2018 Mar 4.
Published in final edited form as: Nat Microbiol. 2017 Sep 4;2(11):1543–1557. doi: 10.1038/s41564-017-0017-2

Figure 6. TRIM23 GTPase activates TBK1 to phosphorylate p62.

Figure 6

a,b, Endogenous p62 phosphorylation at S403 in the WCLs of HEK293T cells that were transfected with si.NT or si.TRIM23 for 48 h and then either mock-treated, infected with mutHSV-1 (MOI 10) for 3 h (a), or treated with rapamycin for 4 h (b), determined by IB with anti-pS403-p62. Knockdown of endogenous TRIM23 was confirmed by IB with anti-TRIM23. c, Endogenous p62 phosphorylation at S403 in the WCLs of HEK293T cells that were transfected with empty vector or FLAG-TRIM23 for 44 h and then mock-treated or treated with BX795 for 4 h, determined by IB with anti-pS403-p62. d, S172 phosphorylation of FLAG-TBK1 in transiently transfected HEK293T cells that co-expressed empty vector or TRIM23-V5, determined by IP with anti-FLAG, followed by IB with anti-pS172-TBK1. e, Laser scanning confocal microscopy analysis of the co-localization of transiently transfected myc-TRIM23 with endogenous phospho-(p)-S172-TBK1 and p62 in HeLa cells, determined by immunostaining with anti-myc (green), anti-pS172-TBK1 (grey), and anti-p62 (red) antibodies at 48 h post-transfection. DAPI, nuclei (blue). Scale bar, 20 µm. f, S172 phosphorylation of HA-TBK1 in HEK293T cells that were co-transfected with empty vector or FLAG-tagged TRIM23 WT or mutants, determined by IP with anti-HA and IB with anti-pS172-TBK1 at 48 h post-transfection. g, Upper panel: TBK1 dimerization assessed by co-purifying HA-TBK1 and GST-TBK1 in transiently transfected HEK293T cells that also co-expressed either empty vector, or FLAG-tagged TRIM23 WT or mutants, determined by IP with anti-GST and IB with anti-HA at 48 h post-transfection. Lower panel: Dimerization of GST-TBK1 and HA-TBK1 was quantified by densitometry, and data shown represent the mean of two independent experiments ± SD. Data are representative of at least two independent experiments (a–g).