HeLa Tet-on cells expressing H-2Kb were nucleofected with 4×100 nM siRNA.
Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, Kb/S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-Kb cells pulsed for 2 h with 10−8 M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2Kd stained with Abs 1, 2 and 3. One of two experiments is shown.