Figure 6: SCIMP drives cytokine selectivity by scaffolding the SFK Lyn for TLR4 phosphorylation. | Nature Communications

Figure 6: SCIMP drives cytokine selectivity by scaffolding the SFK Lyn for TLR4 phosphorylation.

From: SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages

Figure 6

(a) siRNA-mediated SCIMP knockdown in BMM reduces tyrosine phosphorylation of TLR4. After SCIMP silencing (lysates, bottom panels), cells were stimulated for 30 min with LPS, TLR4 was immunoprecipitated and blots were probed for anti-phospho-tyrosine. (b) SCIMP overexpression increases tyrosine phosphorylation of TLR4. RAW264.7 cells stably transfected with empty vector or SCIMP were treated with 30 min with LPS, after which TLR4 was immunoprecipitated and tyrosine phosphorylation was assessed by immunoblotting. (c) IL-12p40 and TNF cytokine production was assessed in primary macrophages (BMM), retrovirally transduced with empty vector (EV), wild-type SCIMP (WT), Y96F SCIMP or W95A SCIMP, at 24 h post-LPS stimulation (mean+s.e.m.). SCIMP protein expression in transduced BMM is shown in the panel on the right. (d) The Src kinase inhibitor SU6656 impairs SCIMP tyrosine phosphorylation and its interaction with TLR4, as assessed by SCIMP immunoprecipitation experiments from lysates of RAW264.7 cells stimulated with LPS for 5 min. (e) Lyn is essential for LPS-induced SCIMP phosphorylation, as well as the interaction between SCIMP and TLR4 as assessed by SCIMP immunoprecipitation from lysates of WT and Lyn−/− BMM stimulated with LPS for 5 min. (f) BMM were pre-treated with SU6656 for 30 min and then stimulated with LPS for 4 h or 8 h. Secreted cytokines (IL-12p40, IL-6 and TNF) were measured by enzyme-linked immunosorbent assay (ELISA; mean+s.e.m.). (g) The effect of SU6656 on cell death in LPS-activated BMM was assessed using the LDH release assay. LDH in culture supernatants was measured as a percentage of total cellular LDH at 8 h post-LPS stimulation from the same samples as in f (mean+s.e.m.). (h) Levels of LPS-induced cytokines (IL-6, IL-12p40 and TNF; ELISA) in culture supernatants from wild type (WT) and Lyn−/− (KO) BMM were assessed at 24 h post-stimulation (mean+s.e.m.). All experiments were repeated at least 3 times, and all graphical data are pooled from at least n=3 independent experiments (mean+s.e.m.). Significance was assessed using one-way ANOVA in c, f and h (*P<0.05 and **P<0.01).

Back to article page