Figure 4.
NAP1 binds to TBK1 during mitosis and is localized to centrosomes. (A and B) Cartoon (A) represents protein domains of full-length (FL) NAP1 and NAP1 lacking the TBK1 binding domain (∆230–270). Cartoon (B) represents protein domains of full length (FL) TBK1 and TBK1 lacking adaptor binding domain (Δ C’ terminal truncation). (C) Representative immunoblots of the pulldowns of transiently expressed GFP-NAP1 or GFP-NAP1 ∆230–270 (lacking TBK1 binding domain) in asynchronous and synchronized mitotic HEK293T cells. Nocodazole was used for cell synchronization. (D) Semiquantitative pTBK1/TBK1 levels after normalization to the pulldown efficiency of GFP-NAP1. Error bars indicate ± SEM; n = 3 independent experiments. (E) Representative immunoblots of the pulldown of HA-FLAG TBK1 in asynchronous and synchronized mitotic TBK1 rescue HeLa cells. Nocodazole was used for synchronization. (F) Semiquantitative NAP1 levels after normalization to the pulldown efficiency of FLAG-TBK1. Error bars indicate ± SEM; n = 3 independent experiments. (G) Representative immunoblots of the pulldown of HA-FLAG TBK1 and HA-FLAG TBK1 Δ C’ (lacking adaptor binding domain) in synchronized mitotic TBK1 rescue HeLa cells. Nocodazole was used for synchronization. (H) Representative immunoblots of the pulldown of endogenous TBK1 in asynchronous and synchronized mitotic DLD-1 cells. Nocodazole was used for cell synchronization. (I) Semiquantitative endogenous NAP1 levels after normalization to the pulldown efficiency of TBK1. Nocodazole was used for cell synchronization. Error bars indicate ± SEM; n = 3 independent experiments. (J) Representative confocal images of HeLa cells transiently expressing EGFP-NAP1 with immunocytochemical detection of p-TBK1 (red). DAPI (blue) was used as a nuclear counterstain. White arrow depicts the area used for line scan analysis in H. Scale bar = 20 μm. (K) Line scan analysis of images in J generated from Nikon Elements software. (L) Representative confocal images of p-TBK1 expression on centrosomes from WT HeLa and NAP1 KO cells. DAPI (blue) was used as a nuclear counterstain (on composite image), α-tubulin for cytoskeleton staining (red), and p-TBK1 S172 conjugated 488 (green). Scale bar = 20 μm. (M and N) Relative intensity of p-TBK1 (M) and area stained by p-TBK1 (N) on the centrosomes of mitotic cells from WT HeLa and NAP1 KO cells. 40–50 mitotic cells per group were quantified from two biological replicates. Error bars indicate ± SEM. One dot = one cell. (O and P) Representative immunoblots (O) and semiquantitative NAP1 levels after normalization (P) of the from the lysates in UT, dTAGv-1 treated, and washed FKBP12F36V-NAP1 DLD-1 cells. 1 μM dTAGv-1 was used for depleting the NAP1 level in the treated sample. For washed samples, NAP1 was first depleted with 2 h of dTAGv-1 treatment and restored after washing and culturing in a normal medium for 4 h. Vinculin was used as a loading control. Error bars indicate ± SEM; n = 3 independent experiments. (Q) Representative confocal images of p-TBK1 expression on centrosomes from UT, dTAGv-1 treated and washed FKBP12F36V-NAP1 DLD-1 cells. DAPI (blue) was used as a nuclear counterstain, α-tubulin for cytoskeleton staining (red), and p-TBK1 S172 conjugated 647 (magenta). Scale bar = 20 μm, insets = 5 μm. (R and S) Relative intensity of pTBK1 (R) and area stained by p-TBK1 (S) on the centrosomes of mitotic cells from UT, dTAGv-1 treated, and washed FKBP12F36V-NAP1 DLD-1 cells. 60–70 mitotic cells per group were quantified from three biological replicates. Error bars indicate ± SEM. One dot = one independent experiment (D, F, I, and P). Unpaired Student’s t test was performed for analysis between two groups, and one-way ANOVA was used for the analysis with three groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Source data are available for this figure: SourceData F4.

NAP1 binds to TBK1 during mitosis and is localized to centrosomes. (A and B) Cartoon (A) represents protein domains of full-length (FL) NAP1 and NAP1 lacking the TBK1 binding domain (∆230–270). Cartoon (B) represents protein domains of full length (FL) TBK1 and TBK1 lacking adaptor binding domain (Δ C’ terminal truncation). (C) Representative immunoblots of the pulldowns of transiently expressed GFP-NAP1 or GFP-NAP1 ∆230–270 (lacking TBK1 binding domain) in asynchronous and synchronized mitotic HEK293T cells. Nocodazole was used for cell synchronization. (D) Semiquantitative pTBK1/TBK1 levels after normalization to the pulldown efficiency of GFP-NAP1. Error bars indicate ± SEM; n = 3 independent experiments. (E) Representative immunoblots of the pulldown of HA-FLAG TBK1 in asynchronous and synchronized mitotic TBK1 rescue HeLa cells. Nocodazole was used for synchronization. (F) Semiquantitative NAP1 levels after normalization to the pulldown efficiency of FLAG-TBK1. Error bars indicate ± SEM; n = 3 independent experiments. (G) Representative immunoblots of the pulldown of HA-FLAG TBK1 and HA-FLAG TBK1 Δ C’ (lacking adaptor binding domain) in synchronized mitotic TBK1 rescue HeLa cells. Nocodazole was used for synchronization. (H) Representative immunoblots of the pulldown of endogenous TBK1 in asynchronous and synchronized mitotic DLD-1 cells. Nocodazole was used for cell synchronization. (I) Semiquantitative endogenous NAP1 levels after normalization to the pulldown efficiency of TBK1. Nocodazole was used for cell synchronization. Error bars indicate ± SEM; n = 3 independent experiments. (J) Representative confocal images of HeLa cells transiently expressing EGFP-NAP1 with immunocytochemical detection of p-TBK1 (red). DAPI (blue) was used as a nuclear counterstain. White arrow depicts the area used for line scan analysis in H. Scale bar = 20 μm. (K) Line scan analysis of images in J generated from Nikon Elements software. (L) Representative confocal images of p-TBK1 expression on centrosomes from WT HeLa and NAP1 KO cells. DAPI (blue) was used as a nuclear counterstain (on composite image), α-tubulin for cytoskeleton staining (red), and p-TBK1 S172 conjugated 488 (green). Scale bar = 20 μm. (M and N) Relative intensity of p-TBK1 (M) and area stained by p-TBK1 (N) on the centrosomes of mitotic cells from WT HeLa and NAP1 KO cells. 40–50 mitotic cells per group were quantified from two biological replicates. Error bars indicate ± SEM. One dot = one cell. (O and P) Representative immunoblots (O) and semiquantitative NAP1 levels after normalization (P) of the from the lysates in UT, dTAGv-1 treated, and washed FKBP12F36V-NAP1 DLD-1 cells. 1 μM dTAGv-1 was used for depleting the NAP1 level in the treated sample. For washed samples, NAP1 was first depleted with 2 h of dTAGv-1 treatment and restored after washing and culturing in a normal medium for 4 h. Vinculin was used as a loading control. Error bars indicate ± SEM; n = 3 independent experiments. (Q) Representative confocal images of p-TBK1 expression on centrosomes from UT, dTAGv-1 treated and washed FKBP12F36V-NAP1 DLD-1 cells. DAPI (blue) was used as a nuclear counterstain, α-tubulin for cytoskeleton staining (red), and p-TBK1 S172 conjugated 647 (magenta). Scale bar = 20 μm, insets = 5 μm. (R and S) Relative intensity of pTBK1 (R) and area stained by p-TBK1 (S) on the centrosomes of mitotic cells from UT, dTAGv-1 treated, and washed FKBP12F36V-NAP1 DLD-1 cells. 60–70 mitotic cells per group were quantified from three biological replicates. Error bars indicate ± SEM. One dot = one independent experiment (D, F, I, and P). Unpaired Student’s t test was performed for analysis between two groups, and one-way ANOVA was used for the analysis with three groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Source data are available for this figure: SourceData F4.

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