Figure 3
Subcellular colocalisation and co-immunoprecipitation of αT2ib and mouse/human TLR2 upon transfection. A, Immunofluorescence analysis by laser scanning confocal microscopy of fixed and permeabilized HEK 293 cells overexpressing mouse TLR2 and transiently transfected with αT2ib expression plasmid. i, Expression of αT2ib analysed using FITC labelled anti-myc antibody, ii, Expression of TLR2 visualized by polyclonal anti-mTLR2 serum and Cy5 labelled goat anti-rabbit antibody; iii, Expression of calnexin visualized by anti-calnexin antibody and Cy3 conjugated goat anti-mouse antibody, iv, Overlay of i and ii, v, Overlay of i, ii, iii. B, 3 × 105 HEK293 cells on a culture plate of 9 cm in diameter were transiently transfected with 5 μg expression plasmid DNA (to overexpress Flag-tagged human TLR2 and/or a myc-tagged intrabody (ib-myc) as indicated) plus 5 μg of empty vector where only one expression plasmid was used. Upon 48 h cells were incubated in 1 ml lysis buffer and either analysed by immunoblotting directly (WCL, 20 ml whole cell lysate) or upon immunoprecipitation (IP) using myc tag-specific antiserum (αmyc) and protein A/G beads (except for *-tagged lane illustrating application of beads only).