Figure 6.

Inhibition of constitutive protein secretion by mutant Nudel. (A) Secretion of hPL-GFP, but not GFP, from HEK293T cells. (B) Controlled expression of Nudel by tetracycline (Tet). To examine the effects of FLAG-Nudel isoforms on hPL-GFP secretion, HEK293T cells were transfected and cultured in the presence (+) or absence (−) of Tet as described in Materials and methods. At the end of the assays, cell lysates were subjected to immunoblotting using anti-FLAG mAb (top), while β-actin served as a loading control (bottom). (C) BSA as the loading control of culture media. 5 μl of each medium collected at the indicated time points was subjected to Coomassie blue staining after SDS-PAGE. (D) Secretion levels of hPL-GFP. 10 μl of each medium was immunoblotted using anti-GFP antibody. (E) The relative secretion levels at 24 h were quantified for comparison. The relative secretion levels in Tet⁻ samples are normalized to that of corresponding Tet⁺ samples (i.e., 100) and presented as the mean ± SD.