Fig 2: GPR37L1 protein has a nuclear localization signal. Nuclear localization signal (NLS) and immunoblots showing the expression of GPR37L1 in the nucleus. (A) Amino acid sequence of GPR37L1 showing the potential bipartite (underlined) and classical monopartite (bold) NLS sequence on the extracellular domain amino terminus, predicted using the nuclear localization signal predictor tools http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi (accessed on 15 March 2019) and http://mleg.cse.sc.edu/seqNLS/ (accessed on 15 March 2019). The signal peptide sequence, the first 25-amino-acid sequence at the amino terminus, is indicated in italics. Extracellular domains are indicated in blue, transmembrane helices in red, and intracellular domains in black. (B–E) hRPTCs were transfected with a plasmid encoding GPR37L1-GFP or with a plasmid pcDNA, an empty vector (mock). (B) Two days after transfection, the expression of GPR37L1-GFP in total (T) or nuclear (N) protein preparations was detected by immunoblotting using an antibody specific to GFP. The same blot was probed sequentially for (C) calnexin, a marker for endoplasmic reticulum; (D) Na+/K+-ATPase, a marker for membrane proteins; or (E) HDAC2, a marker for nuclear proteins. IRDye® 800CW-labeled donkey anti-rabbit IgG (926-32213, Licor, Lincoln, NE, USA) and goat anti-rat IgG (926-32219, Licor, Lincoln, NE) were used for GPR37L1-GFP and calnexin. IRDye® 680RD-labeled donkey anti-mouse IgG (926-68022, Licor, Lincoln, NE, USA) was used for Na+/K+-ATPase and HDAC2. Total or nuclear protein prepared from the hRPTCs transfected with pcDNA (mock), an empty vector, served as control. Molecular size markers (M) are shown in each panel and the sizes of the markers in kDa are indicated to the left of panel B. Positions of the target proteins are indicated by symbols on the right side of the immunoblots: * for GPR37L1-GFP, @ for calnexin, $ for Na+/K+-ATPase, and + for HDAC2. (F) Panel showing the overlay of the Western blots for the four targets using two-color infrared fluorescent protein detection for GPR37L1 and calnexin (green fluorescent signals), and Na+/K+-ATPase and HDAC2 (red fluorescent signals).