Journal: Journal of Animal Science and Biotechnology

Article Title: Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

doi: 10.1186/s40104-021-00585-z

Figure Lengend Snippet: TLR4 overexpression promotes the internalization of E. coli through caveolae-mediated endocytosis. a The activation of proteins involved in caveolae-mediated endocytosis signalling in transgenic (TG) and wild-type (WT) monocytes was detected by immunoblotting at different time points after E. coli infection. b , c The phosphorylation levels of Src and CAV1 were quantified based on immunoblotting. d , e The protein expression levels of Src and CAV1 were quantified based on immunoblotting. All data are presented as the mean ± SD, n ≥ 3; * P < 0.05, ** P < 0.01. GAPDH was used for normalization

Article Snippet: For analysis of the interactions among TLR4, caveolae-dependent endocytosis, and Src signalling in E. coli internalization and elimination, monocytes were pretreated for 2 h with either 2 μmol/L Tak242 (MedChemExpress, Monmouth Junction, USA) —a TLR4 inhibitor, 3 μmol/L filipin—a caveolae-mediated endocytosis inhibitor (MedChemExpress), or 3 μmol/L dasatinib (MedChemExpress) —an Src inhibitor.

Techniques: Over Expression, Activation Assay, Transgenic Assay, Western Blot, Infection, Expressing

Journal: Journal of Animal Science and Biotechnology

Article Title: Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

doi: 10.1186/s40104-021-00585-z

Figure Lengend Snippet: Inhibition of caveolae-mediated endocytosis reduces TLR4-overexpressing monocyte endocytosis and kills E. coli . a Monocytes were pretreated with the control (DMSO) or various inhibitors for 2 h, and after 30 min of E. coli infection (MOI = 10), the protein levels of Src, CAV1, p-Src, and p-CAV1 were determined by western blotting. b – e Quantification of the data in ( a ). GAPDH protein was used for normalization. f – g Flow cytometric analysis of the percentage of monocyte endocytosis of pHrodo- labelled E. coli (MOI = 10) by the TLR4-overexpressing cells pretreated with DMSO or the inhibitors dasatinib (3 μmol/L) or filipin (3 μmol/L) prior to 30 min of infection with pHrodo- labelled E. coli . h The quantity of E. coli internalized within monocytes after 30 min of infection (MOI = 10) was determined based on a colony-forming unit (CFU) count. i After 15 min of incubation with E. coli (MOI = 10), the bacteria that were not internalized were washed away, and the cells were cultured for an additional 15 min in the incubator. The number of E. coli killed in the monocytes was calculated based on the difference in the CFU values at these two time points. All data are presented as the mean ± SD, n ≥ 3; * P < 0.05, ** P < 0.01. DMSO, dimethyl sulfoxide; MOI, multiplicity of infection

Article Snippet: For analysis of the interactions among TLR4, caveolae-dependent endocytosis, and Src signalling in E. coli internalization and elimination, monocytes were pretreated for 2 h with either 2 μmol/L Tak242 (MedChemExpress, Monmouth Junction, USA) —a TLR4 inhibitor, 3 μmol/L filipin—a caveolae-mediated endocytosis inhibitor (MedChemExpress), or 3 μmol/L dasatinib (MedChemExpress) —an Src inhibitor.

Techniques: Inhibition, Infection, Western Blot, Incubation, Cell Culture