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Full-length MYRF is a type-II membrane protein.

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posted on 2013-08-13, 02:44 authored by Zhihua Li, Yungki Park, Edward M. Marcotte

(A) Predicted sequence features of MYRF and sequence diagrams of various MYRF constructs used for experiments. (B) Western blot of HeLa cells transfected with pcDNA3 and 3F-MYRF. (C) The top band of HeLa cells that were transfected with 5M-MYRF-3F has the same electrophoretic mobility as full-length protein products for the same construct that were obtained with an in vitro translation system. (D) Full-length forms of MYRF consist of two closely spaced bands that represent glycosylated and unglycosylated full-length MYRF, respectively (indicated by the two arrows). (E) HeLa cells transfected with 3F-MYRF were disrupted using a Dounce-type homogenizer, and then centrifuged at 200× g for 5 min to obtain a supernatant fraction. It was mixed with 0.1 volume of each of the following chemicals: 5 M NaCl, 1 M Na2CO3 (pH 11), and 10% SDS. After incubation for 20 min at room temperature, mixtures were centrifuged at 20,000× g for 15 min at 4°C to separate supernatant (S) from pellet (P). Calnexin, a known integral membrane protein, served as a control. (F) Membrane topology of GFP-MYRF-3F and 3F-MYRF-L690A-GFP in HeLa cells. When cell membranes were selectively permeated by digitonin, FLAG IF signals of GFP-MYRF-3F could not be detected, indicating that the C-terminus of MYRF is located within the ER lumen. In contrast, FLAG IF signals of 3F-MYRF-L690A-GFP were robustly detected even when cell membranes were selectively permeated by digitonin, indicating that the N-terminus of full-length MYRF is located on the cytoplasmic side of ER membranes. Scale bar, 10 µm.