Other known TBK1 adaptors except NAP1 are not required for TBK1 activation during mitosis. (A–C) qRT-PCR showing relative expression of TANK1 (A), SINTBAD (B), and NAP1 (C) mRNA levels normalized to β-actin in HeLa cells stably expressing shRNAs. n = 3 independent experiments. Error bars ± SD. (D–J) Representative Western blots and semiquantitative analysis of p-TBK1/TBK1 levels normalized to vinculin in asynchronous and synchronized mitotic cells from scramble and cell lines stably expressing TANK (D and E), SINTBAD (F and G), and NAP1 (I and J) shRNA, respectively. Semi-quantitative analysis of p-TBK1/TBK1 levels normalized to vinculin in asynchronous SINTBAD (H) shRNA expressing cells. Nocodazole was used for synchronization. n = 3 independent experiments. Error bars ± SEM. (K and L) Representative Western blot and semiquantitative analysis of p-TBK1/TBK1 levels normalized to vinculin during mitosis in WT and Penta KO HeLa cells lacking NBR1, TAX1BP1, optineurin, NDP52, and p62 in asynchronous and mitotic cells. RO-3306 was used for synchronization. n = 3 independent experiments. Error bars ± SEM. One dot = one independent experiment. Unpaired Student’s t test or one-way ANOVA was performed for all statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. Source data are available for this figure: SourceData F1.