Fig. 2: PGRMC1 binds to mutant POMC and promotes its RTN3-dependent ER-phagic clearance.

a HEK 293 T cells expressing C28F-POMC-FLAG and either empty vector or HA-PGRMC1 were transfected with the indicated siRNAs. Cells were solubilized with RIPA buffer (containing the conventional detergents 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) to generate a “soluble” fraction. The insoluble material was subsequently extracted by 2% SDS and is referred to as the “insoluble” fraction. Both the soluble and insoluble fractions were subjected to SDS-PAGE and immunoblotted as indicated. N = 3 independent experiments. b As in a but using the indicated siRNAs. N = 3 independent experiments. c Quantification of the insoluble C28F-POMC-FLAG level from a, with the protein level relative to the PGRMC1 siRNA-treated condition (lane 2). Data are represented as mean ± SD. N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. From left to right, corresponding p-values are: < 0.028, < 0.0001, < 0.176, < 0.02. d Quantification of the insoluble C28F-POMC-FLAG level from b, with the protein level relative to the PGRMC1 siRNA condition (lane 3). Data are represented as mean ± SD. N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. From left to right, corresponding p-values are: < 0.0001, < 0.272, < 0.003, < 0.006. e HEK 293 T cells expressing C28F-POMC-FLAG and transfected with the indicated siRNAs were treated with cycloheximide for 0, 20, 40, 60 min. The soluble (top) and insoluble (bottom) materials were collected as in a and subject to SDS-PAGE and immunoblotted with the indicated antibodies. N = 3 independent experiments. f HEK 293 T cells were transfected with C28F-POMC-Myc and either FLAG-ERLIN1 or FLAG-PGRMC1. Cells were lysed and immunoprecipitated using a FLAG antibody. Whole-cell lysate (input) and 3xFLAG peptide eluate was subject to SDS-PAGE and immunoblotted as indicated. N = 3 independent experiments. g A schematic of the lyso-IP protocol, as described in method³⁴. h Lyso-IP was performed on HEK 293 T cells transfected with C28F-POMC-Myc and co-transfected with either scramble or PGRMC1 siRNA. Samples were subjected to SDS-PAGE and immunoblotted as indicated. N = 3 independent experiments. i Quantification of C28F-POMC-FLAG in the immunoprecipitated material from h (top panel). N = 3 independent experiments. One-tailed Standard Student’s t-test was used to determine statistical significance. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001. Data are represented as mean ± SD. Source data are provided as a Source Data file. See also Fig. S1.