FIG. 2.

Generation and characterization of FRTL-5 clones stably expressing Flag-tagged N188K. (A) FRTL-5 cells were transfected with Flag-tagged N188K or parental empty vector (pcDNA) as described in Materials and Methods. N188K mRNA levels were measured in two selected clones by real-time PCR. (B) N188K protein levels were measured by immunoprecipitation and Western blot analysis with anti-Flag antibody (upper panel). FRTL-5 clones were transiently transfected with C5-CAT and CMV-Luc as an internal control to assess the effect of N188K on C5-CAT activity (lower panel). The C5-CAT/CMV-Luc ratio in control cells (pcDNA) is arbitrarily reported as 100%. (C) Northern blot analysis with 20 μg of total RNA from mock-transfected FRTL-5 (pcDNA), Cl.1, and Cl.3 cells probed with a cDNA fragment corresponding to rat Tg. (D to F) Western blot analyses of Tg (D), NIS (E), TTF-1 (F), TTF-2 (F), and Pax-8 (F) expression in pcDNA and Cl.1 and Cl.3 cells. The membranes were subsequently stripped and reprobed with antibodies against the indicated antibodies or β-actin and YY-1 for loading control for total or nuclear extracts, respectively. IgG, immunoglobulin G; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.