FIG 8.

The GR interacts with KLF15. Neuro-2A cells were grown to 80% confluence on 100-mm dishes. Cells were cotransfected with plasmids that express KLF15 (1.5 μg) and the GR (2 μg). Cultures were treated with DEX (10 μM) in 2% stripped-serum medium for 4 h (A) before cell lysate was harvested or treated with DEX (B). Whole-cell lysate was prepared, and co-IP studies were performed using the GR or KLF15 antibody as described in Materials and Methods. Following IP with the designated antibody, the GR or KLF15 was detected in the immunoprecipitate by Western blotting (WB). Input lysate (50 μg of protein) was used as a positive control. (C) Neuro-2A cells were cotransfected with plasmids that express KLF15 (0.5 μg), KLF4 (0.5 μg), and the GR (1 μg) as indicated in the figure. Whole-cell lysate was prepared using RIPA buffer, proteins were separated by SDS-PAGE (50 μg in each lane), and GR expression was detected by Western blotting. Cell lysate from nontransfected Neuro-2A cells was used to examine endogenous GR expression. (D) Neuro-2A cells were cotransfected with plasmids that express KLF15 (0.5 μg), KLF4 (0.5 μg), and the GR (1 μg) as indicated on the figure. Whole-cell lysate was prepared using RIPA buffer, proteins (50 μg in each lane) were separated by SDS-PAGE, and Western blot analysis was performed using anti-KLF15. Cell lysate from nontransfected Neuro-2A cells was used to examine endogenous KLF15 expression. (E) As a loading control, tubulin levels were examined. For each lane, 50 μg was loaded. Values on the sides of the blots indicate molecular mass in kilodaltons.