(A) Confirmation of the overexpression of mouse Lass family members in HEK 293T cells by immunoblotting. HEK 293T cells were transfected with pcDNA3-HA (vector), pcDNA3-HA-Lass1, pcDNA3-HA-Lass2, pcDNA3-HA-Lass4, pcDNA3-HA-Lass5 or pcDNA3-HA-Lass6. Total cell lysates (5 μg of protein) were separated on an SDS/10%-polyacrylamide gel and then transferred to membranes. Immunoblotting was then performed using an anti-HA antibody. (B) PNGase F and Endo Hf digestion assay. Denatured samples were prepared from HEK 293T cells transfected with pcDNA3-HA-Lass6, pcDNA3-HA-Lass5 or pcDNA3-HA-Lass2. After incubating with buffer alone, PNGase F or Endo Hf, the cell lysates (5 μg of protein) were separated and subjected to immunoblotting as in (A). An asterisk (*) indicates a glycosylated protein.