Figure 5. Dab2 interacts with clathrin and regulates the association of TLR4 with clathrin.

(a) The shLuc and shDab2-B cells were pre-treated with 7.5 μM CPZ for 30 min followed by LPS stimulation for 18 h. The levels of the indicated cytokines/chemokines present in the culture medium were measured by ELISA. Data represent the mean ± SEM of three independent experiments. (b) The lysates of shLuc and shDab2-B cells were subject to Western blotting using the anti-CHC antibody. The expression of β-actin was used for the control of equal protein loading. The relative expression levels of CHC after normalization by the expression of β-actin are shown. The full Western blot and the corresponding positions of the molecular weight protein markers are presented in Supplementary Fig. S4. (c) The shLuc cells were treated with LPS (100 ng/ml) for the indicated time. The cell lysates were collected for immunoprecipitation of Dab2 using the anti-Dab2 (H-110) antibody. The immuno-precipitated lysates were subject to Western blotting using the anti-CHC antibody. Data represent the mean ± SEM of three independent experiments. The full Western blot and the corresponding positions of the molecular weight protein markers are presented in Supplementary Fig. S4. (d) The shLuc and shDab2-B cells were treated with LPS for 15 and 30 min. Immunofluorescence staining was then performed using the rabbit anti-mouse CHC (red) and biotinylated anti-TLR4 (green) primary antibodies followed by the alexa fluor 555-conjugated donkey anti-rabbit IgG antibody and alexa fluor 488-conjugated streptavidin. The co-localization of TLR4 and CHC was determined by using Zeiss LSM Image Browser Version. Co-localization was determined by use of the co-localization coefficient between 15–30 cells in each group. ns, no significance. *P < 0.05; **P < 0.01; ***P < 0.001, and ****P < 0.0001. Scale bar: 10 μm.