Fig 1: 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.
Fig 2: 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.
Fig 3: Ectopic ACTH expression in LCNEC tissues. (A) Ectopic expression of ACTH in LCNEC tissues as determined by immunohistochemistry. (B) Infiltration of CD3+, CD4+, CD8+, and CD20+ lymphocytes into the tumor. Representative images at low and high magnifications are shown. Scale bar 50 µm. ACTH, adrenocorticotropic hormone; LCNEC, large-cell neuroendocrine carcinoma.
Fig 4: Immunofluorescence staining for detecting autoantibody. Immunofluorescence staining of patient serum and anti-ACTH antibody in the mouse pituitary. (A) Serum from the patient with specifically recognized corticotrophs compared with sera from healthy subjects. Pre-absorption of the patient’s serum with recombinant human POMC protein diminished reactivity to corticotrophs, indicating the presence of circulating anti-POMC antibodies in the serum. (B) Pre-absorption of POMC or ACTH1-39 protein into the serum diminished the signal, whereas pre-absorption of ACTH1-24 or α-MSH protein showed no signal reduction, indicating that ACTH25-39 was the region recognized by the circulating anti-POMC antibody. The representative results are presented in the figure. Scale bar 50 µm. ACTH, adrenocorticotropic hormone; POMC, proopiomelanocortin; α-MSH, alpha-melanocyte-stimulating hormone.
Supplier Page from Abcam for Recombinant Human POMC protein (His tag N-Terminus)