(A) Lysates were prepared from IFN-γ-treated HeLaM, HeLaMKO, HeLaMKOTAPBPRWT, and HeLaMKOTAPBPRC94A cells in 1% digitonin. After preclear, pulldowns were performed with GST/6xHis-tagged exogenous WT human calreticulin (GST-CRTWT), which specifically recognises Glc1Man9GlcNAc2 glycans, or with a CRT variant in which a tyrosine at position 92 had been mutated to alanine (GST-CRTY92A), which disrupts glycan recognition. Western blot analysis was performed for the 6xHis tag, MHC class I HC, tapasin, and TAPBPR on pulldowns and lysates as indicated (see Figure 6—figure supplement 1 for densitometry analysis on these blots and the Endo H-sensitivity status on the GST-CRT-reactive MHC class I molecules). (B) TAP, tapasin, or TAPBPR were isolated by immunoprecipitation from IFN-γ-treated HeLaM, HeLaMKO, HeLaMKOTAPBPRWT, and HeLaMKOTAPBPRC94A cells with or without depletion of UGT1 using shRNA. Western blot analysis was performed for TAP, tapasin, TAPBPR, MHC class I HC, UGT1, or calnexin as a loading control on immunoprecipitates or lysates as indicated, resolved under reducing conditions. (C and D) Quantitative analysis of the MHC class I molecules bound to tapasin and TAPBPR in IFN-γ-treated HeLaMKO, HeLaMKOTAPBPRWT, and HeLaMKOTAPBPRC94A cells from four independent Cy5 experiments using the Amersham WB system (see Figure 6—figure supplement 2 for gel images and further analysis). Scatter dot plots show (C) the ratio of HLA-A68 to HLA-B15 associated with tapasin and (B) the total amount of MHC class I HC bound to TAPBPR as a ratio of the PeTe4 antibody light chain used in the immunoprecitation. *p<0.05, n.s.=not significant based on Mann–Whitney non-parametric, two-tailed tests. KO: knockout; UGT1: UDP-glucose:glycoprotein glucosyltransferase 1; WT: wild-type; IP: immunoprecipitation; WB: western blot.