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. 2023 Feb 17;33(4):273–287. doi: 10.1038/s41422-023-00782-7

Fig. 1. Famsin is secreted after cleavage from Gm11437 by furin.

Fig. 1

a Immunostaining showing the orientation of FLAG-tagged Gm11437 on the cell membrane in Cos7 cells. The signals from anti-Famsin antibody were observed in both unpermeabilized and permeabilized cells, while the signals from anti-FLAG were only detected in permeabilized cells. These results indicate that the N-terminus of Gm11437 has an extracellular orientation. DAPI, 4,6-diamidino-2-phenylindole; TM, transmembrane. Scale bars, 10 μm. b Immunoblots showing the glycosylation status of famsin in HEK293T cells. The protein extracts from culture medium or whole cell lysate were treated with or without deglycosylation enzymes. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. Deglyco, deglycosylation. c Left panel, schematic overview showing the biotin labeling of Gm11437 located at the plasma membrane and its subsequent detection by anti-Famsin antibody. O/N, overnight; IP, immunoprecipitation; IB, immunoblotting. Right panel, immunoblots showing cleavage of famsin from plasma membrane-localized Gm11437 in HEK293T cells. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. d Left panel, schematic diagram showing the subcellular fractionation of HEK293T cells and subsequent detection of famsin by anti-Famsin antibody. Right panel, immunoblots showing cleavage of famsin from cytoplasm-localized Gm11437 in HEK293T cells. The proteins were treated by the deglycosylation mix. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. TCL, total cell lysate; L, light fraction that includes plasma membranes; P, pellet that includes cellular vesicles. ATP1A1 (Na+/K+-ATPase) is a marker of the plasma membrane fraction. e–g Effect of proprotein convertase inhibitors (5 μM, e) or overexpression of different proprotein convertases (f) on Gm11437 cleavage, and effect of furin on cleavage of WT Gm11437 or Gm11437/AA (g) in HEK293T cells. The blue arrowheads indicate full-length Gm11437-FLAG and the red arrowheads indicate famsin. AA, K190A/R191A.