Fig. 3: PGRMC1 uses its luminal domain to recruit cargo.

a Schematic of the topology of PGRMC1, PGRMC2, and the hybrid and mutant variants generated. b HEK 293 T cells were mechanically disrupted and the membranes were pelleted. Membranes treated with or without trypsin (and Triton-X-100) were subject to SDS-PAGE and immunoblotted as indicated. N = 3 independent experiments. c As in b, except HEK293T PGRMC1 knockout (KO) cells transfected with FLAG-PGRMC1 were used. N = 3 independent experiments. d HEK293T cells expressing C28F-POMC-Myc and either FLAG-PGRMC1, FLAG-Hybrid 1 (H1), FLAG-Hybrid 2 (H2), FLAG-Hybrid 3 (H3), or FLAG-PGRMC2 were lysed and FLAG IP was performed as in Fig. 2f, followed by SDS-PAGE and immunoblotting. N = 3 independent experiments. e E. coli purified nonglycosylated POMC-6xHis (Abcam) was incubated with either FLAG-PGRMC1 (purified from HEK 293 T cells) or BSA as a control. Samples were precipitated using magnetic NTA beads, washed extensively, and analyzed by SDS-PAGE with subsequent Coomassie blue staining. ‘M’ indicates protein markers. N = 3 independent experiments. f FLAG IP was performed as in Fig. 2f using the indicated FLAG-tagged constructs as bait. N = 3 independent experiments. g A schematic of PGRMC1 topology indicating the cargo and RTN3-binding regions. Source data are provided as a Source Data file. See also Fig. S2.