Fig. 5: TLR4 mediates RAB10 dephosphorylation during STm invasion.
From: Salmonella exploits LRRK2-dependent plasma membrane dynamics to invade host cells
a and b Representative images (a) and quantifications (b) of phospho-RAB10 (T73) signal measured by western blot in WT Henle 407 cells infected with the indicated STm strains. Cell lysates were collected and immunoblotted for phospho-RAB10 (T73), total RAB10, or ß-actin (loading control) at 30 min p.i. c and d, Representative images (c) and quantifications (d) of phospho-RAB10 (T73) signal measured by western blot in WT Henle 407 cells infected with WT STm, treated with WT STm supernatant (STm S/N), dead WT STm, LPS (100 ng/ml) or ΔinvA/Inv STm (invasion-deficient attenuated strain with inactivation of T3SS-1, and with invasin gene from Yersinia expressed) for 30 min. e and f Representative images (e) and quantifications of (f) phospho-RAB10 (T73) signal measured by western blot. WT, LRRK2 KO and TLR4 KO Henle 407 cells were infected with WT STm and cell lysates were collected at 10 min or 30 min p.i. and immunoblotted for TLR4, phospho-RAB10 (T73), total RAB10, or ß-actin (loading control). g and h Quantifications (g) and representative images (h) of phospho-RAB10 (T73) localization at WT STm invasion sites at the indicated times p.i. WT, LRRK2 KO or TLR4 KO Henle 407 cells were transfected with GFP-RAB10 and then infected with WT STm. Cells were fixed at 10 or 30 min p.i. and stained for phospho-RAB10 (T73) and STm. The cell boundaries in h are depicted by the white outlines. i WT Henle 407 cells and TLR4 KO Henle 407 cells were infected with WT STm and lysed at 2 hours p.i. for CFU counting. Data shown are means ± S.D. for three independent experiments. In g At least 100 invasion sites for each condition in each experiment were scored for the recruitment or the retention of RAB10. P values were calculated using one-way ANOVA (b and d), two-way ANOVA (f and g), or two tailed unpaired t-test (i). P values in d were calculated and labeled between the treated groups with the untreated control. Scale bars, 10 μm. Source data are provided as a Source Data file.
