Extended Data Fig. 5: Additional characterization of the in vitro system and validation of siRNA knockdowns.

a, HA-YIPF1 was translated in RRL in the presence of WT, single- (TMCO1, ΔT; Nicalin, ΔN; CCDC47, ΔC) or double-knockout (TMCO1/Nicalin, ΔTN; TMCO1/CCDC47, ΔTC) rough microsomes at different concentrations (determined by absorbance at 260 nm), isolated by sedimentation, and analyzed by SDS-PAGE and immunoblotting. The percentage of glycosylated YIPF1 is indicated below the gel. b, Plot of the data in (A). c, Membranes prepared from the indicated wild-type, single- or double-knockout HEK293 cell lines were analyzed by SDS-PAGE and immunoblotting. d, Stalled, Flag-tagged YIPF1 constructs truncated at position 183 and 277 were translated in RRL in the presence of wild-type (WT) or single knockout rough microsomes, and the membrane-associated fraction was isolated by sedimentation. Following anti-Flag immunoprecipitation of the digitonin-solubilized membranes, stalled RNCs were isolated by sedimentation and analyzed by SDS-PAGE and immunoblotting. e, Whole cell lysates from HEK293 cells treated with the indicated siRNAs were analyzed by SDS-PAGE and immunoblotting; ‘Ctrl’ is a non-targeting control siRNA.