Journal: Journal of Cell Science
Article Title: ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues
Figure Lengend Snippet: CD8 TMD* is integrated into the membrane with the correct topology. (A) Cartoon of CD8 chimeras used. (B) Stable HeLa cell lines expressing CD8 WT or CD8 TMD* were fixed and labelled for total (permeabilised cells, anti-HA antibodies) and cell surface (intact cells, anti-CD8 antibodies) CD8. Images were collected in parallel using equal exposure times. (C) Cells expressing CD8 TMD* were lysed and subjected to carbonate extraction. Equivalent amounts of supernatant 1 (initial lysate supernatant), 2 and 3 (from sequential carbonate extractions) and the final membrane pellet were analysed by immunoblotting (IB) with antibodies against HA, calnexin (CNX), calrecticulin (CRT) and Hsp70. (D) Lysates of cells expressing CD8 TMD* or CD8 TMD*VN were treated with or without EndoH and analysed by immunoblotting with antibodies against HA, STT3B and actin. Closed arrowhead indicates N-glycosylated protein, open arrowhead indicates deglycosylated protein. (E) Cells expressing CD8 TMD* were permeabilised with digitonin or Triton-X 100 and labelled with antibodies against the extracellular (luminal) domain of CD8 and the cytoplasmic HA epitope. DNA was stained with DAPI. Scale bars: 10 µm.
Article Snippet: Reagents and antibodies Antibodies against CD8 and rabbit HA were from Sigma, antibodies against BAP31, LAMP1, actin, Hsp70 and β-tubulin were from AbCam, anti-ERGIC53 antibody was from Alexis, mouse anti-HA antibody was from Santa Cruz Biotechnology, anti-BiP antibody was from Cell Signaling, anti-CNX and -CRT antibodies for immunoblotting were from Stressgen, anti-CRT antibody for immunofluorescence was from Thermo Scientific and anti-GM130 antibody was from BD Biosciences.
Techniques: Expressing, Staining