JCS009654 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Mutation of the WLM motif amino acids individually to alanine. (A) Cells expressing the Tail19-55 reporter and the W-A, L-A and M-A mutants were analysed using the anti-CD8 uptake assay. Each of the individual mutants was compromised in the ability to relocate to the TGN with the L-A mutant displaying significant cell surface localisation. Bar, 20 μm. (B) The stability of the individual mutants was determined by cycloheximide chase. The W-A mutant was the least stable being degraded with similar kinetics to mutant 7.

• Supplemental Figure S2 -

  Fig. S2. The CD8 reporters are not retained in the endoplasmic reticulum. (A) CD8-CIMPR or CD8-WLM-AAA-expressing cells were grown in 90 mm tissue culture dishes. Cells in one set of dishes were lysed and the CD8 reporter immunoprecipitated to give the total amount of CD8 reporter present (T). Cells in other dishes were incubated with anti-CD8 mAbs for 3 hours at 37°C (post-Golgi) or at 4°C for 30 minutes (cell surface) before lysis. The antibody-CD8 complexes were captured on protein-A-Sepharose. Total (T), post-Golgi (PG) and cell surface (CS) fractions were subjected to SDS-PAGE and western blotting. As not all the CD8 reporter will be accessible to exogenously added antibodies, the lower panel has been enhanced to increase the contrast. The lower migrating (non-O-glycosylated) form (indicated by the arrows) of the CD8-reporter is present in post-Golgi and cell surface fractions and therefore not indicative of retention in the ER. (B) Cells stably expressing either CD8-CIMPR or CD8-WLM-AAA were fixed and labelled with anti-CD8, anti-TGN46 and appropriate secondary antibodies. Neither the CD8-CIMPR nor the CD8-WLM-AAA exhibit labelling patterns consistent with ER localisation. Bar, 20 μm.