Figure 5.

Detection of Nitr9 protein by Western analyses. (a) Western blot analyses of total protein lysates from HEK293T cells transiently transfected with plasmids expressing a Nitr9 isoform and EGFP. Plasmids encode either an endogenous isoform of Nitr9 or a FLAG-tagged Nitr9 as indicated above each lane. The primary antibodies utilized are shown on the left, and the molecular weights of identified bands are shown on the right. The anti-FLAG antibody serves as a positive control for Nitr9 detection, and the anti-GFP antibody indicates transfection efficiency of each plasmid. Note the total protein loaded (bottom) for the Nitr9L isoform is ten times less than that for Nitr9S and Nitr9SS plasmids. Exposure times for chemiluminescence detection are indicated in each panel. (b) Nitr9L and Nitr9S are glycosylated. Western blot analyses of endoglycosidase-treated total protein lysates from HEK293T cells that were transfected with plasmids encoding endogenous Nitr9 isoforms. The anti-Nitr9⁹⁰ antibody recognizes all three Nitr9 isoforms at the predicted size (right). (c) Detection of Nitr9 protein from zebrafish tissues. Western blot analyses of 25 μg of endoglycosidase-treated total protein from zebrafish tissues and HEK293T cells. Note that a nonspecific band (~28 kD) is detected in HEK293T cells as well as in zebrafish kidney and spleen, with high protein loads. Bottom panel indicates loading control using an anti-GAPDH polyclonal antibody.