The generation of soluble CD1e is independent of LAPTM5 expression.

<p>A, B, C) M10 cells co-expressing CD1e and CD1b were transduced with lentiviral vectors expressing a doxycycline inducible anti-LAPTM5 shRNA or anti-GAPDH shRNA. A) The cells were cultured for 2 days in the absence (ni) or presence (i) of doxycycline (1 µg/mL). Knockdown of LAPTM5 expression was checked by RT-PCR (upper panel) and B) by immunofluorescence staining of fixed, permeabilized cells with the anti-LAPTM5 mAb 2.16.1, after treatment (+Baf) or not (−Baf) with bafilomycin. Scale bar: 20 µM. C) The cells were cultured for 2 days in the absence or presence of doxycycline and metabolically pulse chase labeled. CD1e molecules were immunoprecipitated with the mAb 20.6, treated with Endo F and analyzed by SDS-PAGE. mCD1e and sCD1e represent membrane-associated and soluble CD1e molecules, respectively. D) Transfected HEK293 cells expressing CD1e alone or co-expressing LAPTM5-V5 protein were metabolically labeled and chased for 0, 4, 8 and 18 hours. CD1e molecules were immunoprecipitated from Triton X100 lysates with the mAb 20.6, treated with Endo H or Endo F and analyzed by SDS-PAGE. The position of the Endo H-sensitive membrane-associated CD1e molecules is indicated by a star, those of the Endo H-resistant soluble CD1e molecules by arrows and “R".