CUL3 ^KLHL12 is needed for ER-phagy and the ubiquitination of RTN3L. (A) YFP-RTN3L in LAMP1-mCherry structures in siCtrl and siRNA-treated cells incubated with Baf (4 h). Representative images are shown on the left and in Fig. S8 A. The control for each condition was set to 1.0. (B) RTN3L coprecipitates with KLHL12, but not CALCOCO1. Immunoprecipitations were performed as in Fig. 1 A only the precipitates were eluted using HA peptide as described in the methods. Left, input is 2% of the lysate. Right, equal KLHL12 inputs were used for the immunoprecipitates. (C) Same as B, except the immunoprecipitates were eluted in sample buffer. 4.8 x more PEF1 was precipitated in the tagged sample compared to the untagged control. Input is 2% of the lysate. Equal amounts of PEF1 and SEC24C were used for the immunoprecipitates (see Fig. S8 B). (D) Left, high (asterisks) and low (brackets) molecular weight forms of ubiquitinated RTN3L were detected with ubiquitin antibody. Right, cells were untreated (DMSO) or treated with Baf or Torin 2 + Baf (4 h). (E) The ubiquitination of RTN3L is dependent on KLHL12 and CUL3. Error bars represent SEM, n = 3–6 independent experiments. The results in A were quantified from 59 to 154 cells. NS: not significant (P ≥ 0.05), * (P < 0.05), **(P < 0.01), *** (P < 0.001), Student’s unpaired t test. Source data are available for this figure: SourceData F3.