Related to Fig. 4 . The localization of RTN3L puncta to three-way junctions is not dependent on CUL3^KLHL12. (A) Left, the level of RTN3L, but not SEC24C or KLHL12, increases in CUL3-depleted U2OS cells. Western blot analysis was performed with lysates from Ctrl and CUL3-depleted cells. Quantitation of the blot revealed there was approximately a 1.5 times increase of RTN3L in siCUL3-treated cells compared with siCtrl cells, when the RTN3L levels were normalized to GAPDH. Right, western blot analysis was performed with lysates prepared from cells that were treated with DMSO or Baf for 3.5 h. The LC3-II/I ratio is reported in the figure. The data was normalized to GAPDH. (B) Left, cells were transfected with LNPK-GFP and mCherry-RTN3L. Arrowheads mark colocalizing puncta. Middle, quantification of percent LNPK puncta colocalizing with RTN3L puncta, and average LNPK puncta/cell (right) in siCtrl and siCUL3-treated cells. (C) Representative images of the colocalization of mCherry-RTN3L puncta with YFP-SEC24C puncta in Ctrl and CUL3-depleted cells that were treated with Torin 2 for 3.5 h. Arrowheads mark colocalizing puncta. Error bars in B and C represent SEM, n = 3 independent experiments. The results were quantified from 42 to 43 cells in B, and 38–41 cells in C. NS: not significant (P ≥ 0.05), Student’s unpaired t test. Source data are available for this figure: SourceData FS10.