Non-glycosylated TEM8 is an ER quality control and ERAD substrate.

A) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated or not with MG132, an inhibitor of the proteasome or Bafilomycin A1, a drug preventing endosomal acidification and thus lysosomal degradation. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and TEM8-HA. B) HEK cells stably expressing CMG2 under the control of a tetracycline inducible promotor were induced for 24h with 0.1μg/ml doxycycline. Cells were treated or not with MG132 or Bafilomycin A1. Immunoprecipitates against CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and CMG2-V5. C) HeLa cells were treated or not with tunicamycin, an antibiotic blocking the co-translational transfer of glycan sidechains in the ER by blocking the oligosaccharyltransferase (OST) for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for TEM8 or Calnexin as a negative control. D) RpeI cells were treated or not with tunicamycin for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for CMG2 or Calnexin as a negative control.