Figure 1: Immune-restricted SCIMP is a TLR4-associated cell surface protein enriched in microdomains.

(a) mRNA expression of SCIMP was assessed in the indicated fibroblast, lymphoid and myeloid cell lines. Data represents mean+s.e.m. (n=3). (b) Immunostaining of endogenous SCIMP in LPS-activated RAW264.7 cells (green) on filopodia; cells were co-stained with phalloidin (red) and 4′,6-Diamidino-2′-phenylindole dihydrochloride (DAPI; blue). (c) Fluorescent imaging of LPS-treated (30 min) RAW264.7 cells transiently transfected with SCIMP-GFP (green). The cells were co-stained with DAPI (blue). (d) Immunogold labelling on cryo-EM sections of RAW264.7 cells stably expressing GFP-SCIMP. GFP labelling ruffles at the cell surface. N=nucleus. Gold particles on ruffle or filopodia membranes versus other stretches of plasma membrane were counted (n=5 cells). Significance was assessed using the Student’s t-test (**P<0.01). (e) GST-SCIMP-T1 coupled to GSH-Sepharose was used for pull-downs from LPS-activated RAW264.7 cell extracts. Bound proteins were eluted by a protease cleavage elution method and separated by SDS–PAGE. Excised bands were identified by liquid chromatography and mass spectrometry (LC/MS/MS). A band at ∼100 kDa, absent from the GST control, was identified as TLR4. (f) List of the top hits from the LC/MS/MS analysis of SCIMP-GST pull downs. Data in a–f are representative of at least three independent experiments. Scale bars in b–d represent 10 μm, 20 μm and 10 nm, respectively.