Fig 2: Lack of catalytic domain glycosylation impairs TMPRSS13 cell surface localization. Twenty-four hours after seeding onto glass coverslips, HEK293T cells were transfected for 48 h with (A) WT-TMPRSS13 (T13)-V5 plus empty vector (EV), (B) WT-TMPRSS13-V5 plus HAI-2-EYFP, (C) S506A-TMPRSS13-V5 plus EV, (D) N400Q/N440Q-TMPRSS13-V5 plus EV, (E) N400Q/N440Q-TMPRSS13-V5 plus HAI-2-EYFP, or (F) EV plus EV. Cells were fixed (no permeabilization), incubated overnight with anti-V5 antibody, and analyzed by confocal microscopy. Nuclei (DAPI) (blue, A–F), TMPRSS13-V5 (red, A–F), HAI-2 (green, B and E). Merged images are shown in panels on the right.

Fig 3: Expression analysis of matriptase, HAI-1, HAI-2, and prostasin in B cancer cells by reverse-transcription/qPCR (a,b), western blotting (c–e), and flow cytometry (f). (a) Bar graph of relative mRNA expression levels of matriptase (Mat), HAI-1, HAI-2, prostasin (Pro) in Daudi (n = 4), Namalwa (n = 5), Ramos (n = 3), Raji (n = 3), JeKo-1 (n = 3), and RS4;11 cells (n = 2) using GAPDH as the reference. The prostasin bars do not appear in the bar graph, as the actual qPCR readouts were registered as “N/A” by the instrument. (b) Bar graph of mRNA quantity ratio of HAI-2 to matriptase after normalization with the GAPDH level in each cell line in (a). (c) Western blotting images of matriptase (Ab: A300-221A), HAI-2, and GAPDH. Twenty micrograms of total protein from the cell lysate of each individual culture (including 2 repeats) were analyzed. Daudi, lanes 1–3; Namalwa, lanes 4–6; Ramos, lanes 7–9. Top panel, matriptase (Mat); middle panel, HAI-2; bottom panel, GAPDH. (d) Densitometry bar graph of relative protein quantities of matriptase and HAI-2 using GAPDH as the reference. (e) The quantitative ratio of HAI-2 to matriptase in each cell line. (f) Flow cytometry histogram of matriptase expression evaluation in Ramos cells. The Ramos cells (4 × 105) were labeled with the matriptase antibody as described in the Materials and Methods section. The matriptase-positive cells are shown in the PE-A subset (blue peak). Cells without the matriptase antibody labeling (red peak) were not detected in the PE-A subset and were used as the gating control.

Fig 4: Glycosylation deficiency causes ER retention of TMPRSS13. Twenty-four hours after seeding onto glass coverslips, HEK293T cells were transfected for 48 h with (A) WT-TMPRSS13 (T13)-V5 plus empty vector (EV), (B) WT-TMPRSS13-V5 plus HAI-2-EYFP, (C) S506A-TMPRSS13-V5 plus EV, (D) N400Q/N440Q-TMPRSS13-V5 plus EV, (E) N400Q/N440Q-TMPRSS13-V5 plus HAI-2-EYFP, or (F) EV plus EV. Cells were fixed, permeabilized, and incubated overnight with anti-V5 to detect TMPRSS13 and anti-KDEL to detect endogenous KDEL. Nuclei (DAPI) (blue, A–F), TMPRSS13-V5 (red, A–F), HAI-2 (green, B and E), KDEL (cyan, A–F). Merged images of TMPRSS13/KDEL are shown in panels on the right.