Figure 5.

MT2 cleaves Hjv and mildly reduces its cell-surface localization. A, diagram of N-terminal-tagged mouse Hjv with the potential cleavages sites by MT2, furin, and PI-PLC, as well as the antibodies used for Western blot. B, MT2, but not S762A-MT2 or MT2-mask, cleaves Hjv in HEK293 cells. HEK293 cells in 12-well plates were co-transfected with an equal amount of pCMV9-Hjv (2 μg), increasing amount of pCMV6-MT2, S762A-MT2, or MT2-mask (0, 0.5, 1.0, and 2.0 μg), and decreasing amount of pEGFP-N1 (2.0, 1.5, 1.0, 0.5, and 0 μg). All cells were transfected with equal amounts of total plasmid DNA. Fresh medium was changed at 24 h post-transfection. After another 24 h of incubation, MT2 and Hjv in ∼150 μg of cell lysate proteins (L) were immunodetected by using anti-FLAG antibody. β-Actin and EGFP were detected by anti-β-actin and GFP antibodies, respectively. Hjv in ∼600 μl of CM was immunodetected by both anti-FLAG and anti-HJV antibodies. HEK293 cells (Ctrl) were included as a negative control for Hjv and MT2. Two MT2 images with different exposure times are presented. C, expression of MT2 mildly decreases cell-surface Hjv. HEK293 cells were co-transfected with pCMV9-Hjv and an equal amount of pEGFP, pCMV6-MT2, or S762A-MT2 plasmid DNA. At about 48 h after transfection, cell-surface proteins were biotinylated at 4 °C, followed by pull-down of the biotinylated proteins using streptavidin-agarose beads. The eluted cell-surface proteins and about 10% of input lysate were subjected to SDS-PAGE and immunodetection of Hjv, MT2, Na⁺K⁺-ATPase (NaKATPase), β-actin, and EGFP using specific antibodies. D, quantification of Hjv bands in C. The intensities of full-length Hjv bands in cell lysate input and cell-surface eluates, as well as the furin-cleaved Hjv band in CM migrating at ∼45 kDa, were quantified by using an Alexa Fluor 800 goat anti-mouse secondary antibody and an Odyssey Infrared Imaging System (Li-Cor). Only the data with and without MT2 were presented. Pair and two-tailed t test was used to calculate the significant difference between two groups. Results are from five independent experiments. E, a small proportion of MT2 is localized on cell surface. HEK293 cells were transfected with pCMV6-MT2, S762A-MT2, or MT2-mask, followed by biotinylation of cell-surface proteins and Western blot analysis by using specific antibodies. * denotes nonspecific band. All experiments were repeated at least three times (technical replicate = 1; independent biological replicates ≥3) with consistent results.