Related to Fig. 1 . SAR1:T39N does not block the delivery of misfolded Akita to lysosomes. (A) SEC24C coprecipitates with RTN3L. Left, inputs showing that equal amounts of SEC24C were used for the IP in Fig. 1 A. Right, same as Fig. 1 A only the samples were blotted for SEC23A and SEC24A. (B) RTN3 depletion experiments performed in this study were done with two different siRNAs, siRTN3 (directed at a site in the reticulon domain, see details in the Materials and methods) and siRTN3L (directed at two sites in the long domain, see details in the Materials and methods). The same results were obtained with both siRNAs. Western blot analysis was performed using cell lysates treated with siRTN3 and siRTN3L. GAPDH was used as a loading control. (C) RNAi resistant mCherry-RTN3L suppresses the accumulation of large Akita-sfGFP puncta (≥0.5 µm²) in RTN3-depleted cells. Representative images (left) and quantitation of cells with large Akita puncta (right) are shown. Arrows mark large puncta. (D) Western blot analysis was performed using cells treated with siRTN3L, siSEC24C, siCALCOCO1 (left), and siSEC31A (right). GAPDH was used as a loading control. The depletion of RTN3L did not alter the expression of SEC24C or CALCOCO1. (E) Quantitation of Akita-sfGFP puncta in LAMP1-mCherry structures in DMSO treated or Baf treated (3.5 h) cells that were untransfected (Ctrl) or transfected with SAR1:T39N. Arrowheads mark Akita puncta delivered to lysosomes. The data was quantitated as described in the methods. (F) siCtrl, SAR1:T39N, and siRNA-treated cells were transfected with Proinsulin-sfGFP. The percent cells with Proinsulin-sfGFP puncta (≥0.35 µm²) are reported. Arrows mark puncta that were quantitated. Error bars in C, E, and F represent SEM, n = 3 independent experiments. The results were quantified from 29 to 53 cells in C, 65–76 cells in E, and 74–149 cells in F. NS: not significant (P ≥ 0.05), * (P < 0.05), **(P < 0.01), Student’s unpaired t test. Source data are available for this figure: SourceData FS1.