Establishing an inducible Nef system in HeLa cells.
a, Schematic representation of the Nef-ER construct and its mode of action. b, Western blot of stably and transiently transfected cells, labelled with anti-Nef and with anti-clathrin as a loading control. Clone 25 and clone 28 are two different cell lines stably expressing Nef-ER. The cells in the Nef WT trans and Nef-ER trans lanes were transiently transfected with wild-type Nef and with Nef-ER, respectively. c, Cells stably expressing Nef-ER were either left untreated or treated with 100 nM 4HT for 48 hours, then labelled with an antibody against Nef. The label is diffuse and cytosolic in the absence of the drug, but becomes membrane-associated after the drug is added. d, Surface HLA-A2 in cells treated with different concentrations of 4HT for 48 hours, either continuously at 37°C or with a shift to 26°C after 24 hours. e, Surface HLA-A2 in 4-HT-treated and control cells, with or without various siRNA knockdowns.