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. 2007 Mar 2;3(3):e29. doi: 10.1371/journal.ppat.0030029

Figure 4. TKB1 Knockdown in Epithelial Cells Phenocopies TBK1 Deficiency in MEFs.

Figure 4

(A) HeLa cells were transfected with the indicated siRNA, and knockdown at 72 h p.i. was determined by Western blot analysis using an anti-TBK1 antibody: lane (1) no siRNA, lane (2) scrambled siRNA, lane (3) tbk1 siRNA (inset). Blots were re-probed with anti-actin antibody as a loading control. HeLa cells were transfected with tbk1 or control siRNA and then infected by Salmonella to measure intracellular growth over time; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a one-tailed Student t-test comparing tbk1 siRNA with control siRNA-treated cells at each time point.

(B) HeLa cells transfected with tbk1-specific or control siRNA were infected with Salmonella-GFP. Samples were fixed at 4 h p.i., stained with anti-ubiquitin antibody followed by a TRITC-labeled secondary antibody (red), and analyzed by confocal microscopy. The experiment shown is representative of four independent experiments.

(C) Quantitative analysis of cytosolic GFP-Salmonella was performed in infected HeLa cells left untreated or transfected with tbk1, gapdh, or control siRNA. Percent ubiquitin-associated represents the number of bacteria out of 150 counted per experiment that colocalized with ubiquitin at 4 h p.i. from three independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test comparing all samples to untreated cells.

(D) HeLa cells transfected with tbk1-specific or control siRNA were infected for 1 h with the indicated bacteria, fixed at 4 h p.i., and stained with anti-LAMP1 antibody. Percent of LAMP-1-associated bacteria was determined by counting the number of bacteria out of 150 per experiment that colocalized with LAMP-1 as observed by confocal microscopy. The experiment shown is representative of at least two independent experiments; * and ** denote p ≤ 0.05 and 0.001, respectively, according to a two-tailed Student t-test comparing tbk1 siRNA-transfected cells to control-transfected cells for each bacterial strain.