Fig. 3.

CD8^TMD* is an ERAD substrate. (A) Measurement of protein degradation by cycloheximide chase assays. Cells expressing CD8^WT or CD8^TMD* were chased by incubation with cycloheximide for up to 4 h. Cell lysates were analysed by immunoblotting (IB) with anti-HA and anti-actin antibodies, followed by secondary antibodies labelled with infrared fluorophores. (B) The anti-HA antibody signal normalised relative to the anti-actin signal is expressed as a percentage of that present at the start of the chase. Graphs represent the mean±s.e.m. of three independent experiments. (C) Cells expressing CD8^TMD* were left untreated or treated with leupeptin and pepstatin A (L/P) or PSII for 2 h, then chased with cycloheximide in the continued presence of inhibitors for up to 2 h, then analysed as in A. (D) Protein levels were quantified as in B. (E) Cells were induced with tetracycline (tet) to express CD8^TMD* or left uninduced, treated with or without PSII for 8 h, then lysed and the CD8 immunoprecipitated (IP) with anti-HA antibodies. Samples were analysed by immunoblotting with anti-ubiquitin and anti-HA antibodies. *HC, IgG heavy chain; Ub_n, polyubiquitylated proteins; T, 5% of the total input; IP, immunoprecipitated sample; S, supernatant after immunoprecipitation. (F) Cells expressing CD8^TMD* were left untreated, or treated with leupeptin and pepstatin (L/P) or chloroquine for the indicated time. Cell lysates were analysed by immunoblotting with anti-HA and anti-α-tubulin antibodies. u, i and m indicate the unglycosylated precursor, an initially glycosylated intermediate, and the mature glycoform of CD8, respectively.