The HEK 293 cells were transfected with wt FLAG-Bsep, G982R and ΔGly. Untransfected HEK cells were used as a negative control (mock). The wt and mutant FLAG-Bsep were isolated by immunoprecipitation (IP) and eluted from Protein G agarose with a three-FLAG peptide. Any co-precipitated protein was probed by immunoblotting. The probed proteins include p97, calnexin, Hsc70, HRD1, HERP, CHIP and derlin-1. An aliquot of cell lysate was also analyzed, which corresponds to the one tenth of the material used for IP. ECL was used to visualize the protein of interest.