Related to Fig. 3 . The ubiquitination of RTN3L increases in the presence of Baf. (A) Left, CUL3 blot showing that the neddylation of CUL3 is blocked 4 h after treatment with 1 µM MLN4924. Right, same as Fig. 3 E only the sample in lane 4 was treated for 4 h with Baf with 1 µM MLN4924. (B) Same as Fig. 3 D only the sample was treated with 10 µM TAK243 for 4 h. (C) U2OS cells were treated for 4 h with 10 μM MG132 before lysates were prepared for western blot analysis. GAPDH was used as a loading control. (D) Membranes were prepared from control, siCUL3 and siSEC24C-treated cells and crosslinked with the indicated concentration of EGS as described in the methods. (E) Top, the position of the known RTN3L ubiquitination sites are shown. Five of the nine sites are in the C-terminus of the RHD. Bottom, the five sites shown are predicted to be at the end of the second hairpin in the RHD. Right, images of mCherry-RTN3L and mCherry-RTN3L-5KR. The mCherry-RTN3L-5KR mutant contains five mutations (K977R, K979R, K995R, K999R and K1003R) that were found to disrupt the tubular ER network. (F) Representative control images of LNPK-GFP puncta in LAMP1-mCherry structures in siCUL3 and siKLHL12-treated cells for the data shown in Fig. 4 A. (G) Representative control images of Akita-sfGFP puncta in LAMP1-mCherry structures in siCtrl and siRNA-treated cells for the data shown in Fig. 4 B. The siCtrl was also treated with Baf + MRT68921 for 3.5 h to block autophagosome maturation.