Fig. 1: VLRF in the lamprey is a member of the VLR family.

a Germline VLRF gene in the sea lamprey. b Isolation of mature (M) and germline (GL) VLRF sequences from electrophoretic bands (n = 3). c Schematic of the mature VLRF sequence. d Alignment of representative mature VLRA, VLRB, VLRC, VLRD, VLRE, and VLRF sequences in the sea lamprey. Conserved cysteine residues in LRRNT and LRRCT are highlighted in yellow. e Number of LRRV modules in VLRA, VLRB, VLRC, and VLRF repertoires in the sea lamprey. The ending-LRRVe module is included in the number of LRRVs of a mature VLR sequence. 100 mature sequences of each VLR isotype are analyzed. f Predicted 3D structure of VLRF, highlighting the protruding loop in the LRRCT region of this assembly (GenBank accession no. PQ159758). The LRRNT and LRR1 regions are shown in blue, LRRVs are shown in green, while the CP and LRRCT regions are represented in red. g Analysis of the length of the HVR in the LRRCT domain. The trends in the HVR length distribution of VLRB are distinct from those of VLRA, VLRC and VLRF. h A phylogenetic tree constructed using five representative mature sequences for each VLR isotype in the sea lamprey. Bootstrap support values are provided for interior branches. The scale bar represents sequence substitution per site. i Prediction of the transmembrane domain (TM) in VLRF. j Histograms showing anti-HA staining on transfected cells detected by flow cytometry. HEK 293 cells were transfected with an Empty vector, HA epitope-tagged VLRF clone 1 or VLRF clone 2. k Whole cell lysates from transfected HEK 293 cells were analyzed by immunoblotting for the presence of HA epitope (VLRF) and beta actin loading control. Brackets indicate the expected bands for VLRFs with three (clone 1) and four (clone 2) LRRV domains. Each blot is a representative of two independent experiments.