Figure 2.

Flagellin induces co-localization of asialoGM1 and GM1 with TLR5. (A) NCIH292 cells exposed to flagellin (Flgn, 10 μg/ml, 30 min) were lysed and immunoprecipitated with either α-ASGM1 antibody (top panel) or α-TLR5 antibody (bottom panel) and then immunoblotted with α-TLR5 antibody. Increased association between ASGM1 and TLR5 occurred in the presence of flagellin (A, top panel), while expression of TLR5 in the same lysates remained equivalent (A, bottom panel). (B) NCIH292 cells plated on Lab-Tek II slide wells were incubated on ice in serum-free RPMI medium containing 600nM Bodipy GM1 (mimicking ASGM1; Molecular Probes) for 30 min before the addition of flagellin. Cells were maintained at either 4°C (B, top panel) or 37°C (B, bottom panel) for 15 min and then exposed to flagellin (10 μg/ml, 10 min). Cells were fixed with 3% paraformaldehyde for 20 min at room temperature and immunostained using rabbit polyclonal α-TLR5 antibody (1:200) with an α-rabbit cy3 secondary antibody (1:2,000). Nuclei were stained blue using Dapi in Vectashield Mounting medium (Vector Laboratories, Inc.). Co-localization of TLR5 and GM1 (far right panel) is apparent in cells maintained at 37°C but not those maintained at 4°C. Staining with isotype control rabbit IgG was negative (not shown).