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. 2022 Feb 9;96(3):e01949-21. doi: 10.1128/jvi.01949-21

FIG 1.

FIG 1

Hamster CADM1 having exon 8 and CADM2 having exon 9 do not induce membrane fusion. (A) Fusion assay with human, mouse, and hamster CADM1 (left) and CADM2 (right). pCA7 expression plasmids encoding the H protein, the F(T461I) protein, and EGFP with pCA7 encoding human, mouse, or hamster CADM1/2 or pCA7 alone as a negative control (NC) were used to transfect 293FT cells. Cells were observed by fluorescence microscopy 30 h after transfection. Exon organizations of CADM1 and CADM2 used in this experiment are also shown. The numbers above rectangles are exon numbers. The numbering of the exons follows previous studies (39, 44–46). The exons present in all isoforms are indicated by gray rectangles, and the exons that are present in only some isoforms are indicated by red, yellow, or magenta rectangles. Bar = 250 μm. (B) Fusion assay with different isoforms of hamster CADM1 (left) and CADM2 (right). pCA7 expression plasmids encoding the H protein, the F(T461I) protein, and EGFP with pCA7 encoding one of the hamster CADM1/2 isoforms or pCA7 alone (NC) were used to transfect 293FT cells. Cells were observed by fluorescence microscopy 30 h after transfection. Bar = 250 μm. (C and D) DSP assay with human (h), mouse (m), and hamster (ham) CADM1 (C) and CADM2 (D) isoforms. pCA7 expression plasmids encoding the H protein and the F(T461I) protein with pCA7 encoding one of the CADM1/2 isoforms or pCA7 alone were used to transfect cocultured 293FT/DSP1 and 293FT/DSP2 cells. Renilla luciferase activity was measured 24 h after transfection (n = 3, means ± standard deviations [SD]). (E and F) Cell surface biotinylation assay with hamster CADM1 (E) and CADM2 (F) isoforms. pCA7 encoding E-cadherin (a cell surface protein control) with pCA7 encoding Flag-tagged EGFP (an intracellular protein control), one of the Flag-tagged hamster CADM1/2 isoforms, or pCA7 alone was used to transfect 293FT cells. Precipitates of biotinylated cell surface proteins and cell lysates were detected by Western blotting using anti-E-cadherin (top) or anti-Flag Ab (bottom).