Guanylate-Binding Protein 1 Regulates Infection-Induced Autophagy through TBK1 Phosphorylation

Figure 5

GBP1 interacts with TBK1 and regulates its phosphorylation. (a) Western blotting image of phosphorylated TBK1 (pTBK1) in HeLa WT and GBP1-KO cells during GAS infection. Cells were collected at the indicated time point. (b) Quantification of the pTBK1/TBK1 ratio in (a). Data are presented as the from three independent experiments. Asterisks indicate statistically significant differences () as determined by two-tailed Student’s -test. (c) pTBK1 recruitment to GAS in HeLa WT and GBP1-KO cells. Cells were transfected with the indicated plasmids and then infected with GAS for 4 h. pTBK1 was colocalized with the bacteria (white outlined arrowhead) and GBP1 (white arrowhead). All images are representative of three independent experiments. Scale bar: 10 μm. (d) Percentage of cells containing pTBK1-positive GAS. Data are presented as the from three independent experiments (100 infected cells were examined in each experiment, counted manually using fluorescence microscopy). Asterisks indicate statistically significant differences () as determined by two-tailed Student’s -test. (e) Immunoprecipitation of GFP-GBP1 intact or GBP1 mutants and FLAG-TBK1. Cells cotransfected with the indicated plasmids were subjected to immunoprecipitation using GFP-Trap. The immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting with anti-GFP and anti-FLAG antibodies. (f) Schematic representation of intact GBP1 and mutants with deletion of different domains of GBP1. (g) Immunoprecipitation of GFP-GBP1 intact or mutants with deletion of GBP1 domains and FLAG-TBK1. Cells cotransfected with the indicated plasmids were subjected to immunoprecipitation using GFP-Trap. The immunoprecipitated proteins and total cell lysates were analyzed by immunoblotting with anti-GFP and anti-FLAG antibodies.

(a)

(b)

(c)

(d)

(e)

(f)

(g)