Fig. 4: TBK1 phosphorylates TRIM23 at S39.

a S39 phosphorylation of TRIM23-FLAG in HEK293T cells that were co-transfected with the indicated siRNAs, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-p-S39-TRIM23. b S39 phosphorylation of TRIM23-FLAG in HEK293T cells that co-expressed either empty vector (–) or increasing amounts of HA-TBK1 or HA-IKKβ, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-p-S39-TRIM23. c S39 phosphorylation of TRIM23-FLAG in HEK293T cells that co-expressed the indicated HA-tagged TBK1 constructs, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-p-S39-TRIM23. d Top: In vitro kinase assay assessing S39 phosphorylation of purified GST-TRIM23 upon co-incubation with purified GST-TBK1, determined by IB with anti-p-S39-TRIM23, anti-TRIM23 and anti-TBK1. Bottom: Densitometric analysis of the p-S39-TRIM23 signal intensities, normalized to the respective TRIM23 protein abundances, for the data shown on top. e S39 phosphorylation and ubiquitination of TRIM23-FLAG in transiently transfected HEK293T cells that were  treated with either DMSO or increasing doses of BX795 (0.5, 1 and 2 µM) for 4 h, determined at 48 h post-transfection by IP with anti-FLAG and IB with the indicated antibodies. f Endogenous TRIM23 S39 phosphorylation in HDFs that were either mock-treated or infected with mutHSV-1 (MOI 5) for 12 h and then treated for 4 h with either DMSO or BX795 (5 µM), determined by immunostaining with the indicated antibodies and confocal microscopy analysis. DAPI, nuclei (blue). Scale bar, 20 µm. g Quantification of p-S39-TRIM23-positive cells for the data shown in (f). h Top: Representative confocal microscopy images showing the colocalization of endogenous p-S39-TRIM23 with p-S172-TBK1 in HDFs that were either mock-treated or infected with WT HSV-1 or mutHSV-1 (MOI 0.3 for both) for 8 h, determined by Proximity Ligation Assay (PLA). Bottom: Quantification of the PLA signals for the data shown on top. PLA signal (magenta), HSV-1 ICP27 (green), nuclei (DAPI, blue). Scale bar, 20 μm. i S39 phosphorylation of TRIM23-FLAG in transiently transfected TBK1 KO single cell clones (Cl-1 and Cl-2) and WT HEK293T cells, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-pS39-TRIM23. j S39 phosphorylation of FLAG-tagged TRIM23 WT or S39A in TBK1 KO HEK293T cells that were co-transfected with either empty vector (–) or HA-TBK1, determined at 48 h post-transfection by IP with anti-FLAG and IB with anti-pS39-TRIM23. k GFP-LC3B puncta formation in TBK1 KO and WT A549 cells that were transiently transfected with GFP-LC3B (green) and either empty vector or the indicated FLAG-tagged TRIM23 constructs (red), assessed at 48 h post-transfection by immunostaining with anti-FLAG followed by confocal microscopy analysis. DAPI, nuclei (blue), scale bar, 20 µm. l Quantification of GFP-LC3B puncta for the data shown in (k). All data (a–l) are representative of at least two independent experiments (mean ± SD of n = 4 blots (d), n = 4 biological replicates (g); mean ± SEM of n  = 49, 48, 52, 51 and 49 cells respectively for the data plots from left to right (h); n  = 26 cells (l). **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed Student’s t test (d, g), two-tailed Student’s t test with Welch’s correction (h, l). NS not significant. Source data are provided as a Source Data file.