Figure 2.

GADD45a induces Rag1 transcription by a MEKK4- and p38-dependent pathway. (a) Flow cytometry of GFP expression in AMuLV-transformed Rag1-GFP cells infected with retrovirus expressing ER-GADD45a (Thy-1.1⁺; solid lines) and treated for 18 h with vehicle control (Veh) or tamoxifen (Tamox). Filled histograms, uninfected cells (Thy-1.1⁻) in the same culture. Numbers above bracketed lines indicate percent GFP⁺ cells in the infected population (top number) and uninfected population (bottom number in parenthesis). (b) GFP expression in ER-GADD45a-expressing cells infected with retrovirus encoded shRNA (above plots) and treated with tamoxifen for 24 h, then labeled with anti-hCD2 and gated for infected cells (expressing shRNA; solid lines) or uninfected cells (not expressing shRNA; filled histograms). Numbers above bracketed lines indicate percent GFP⁺ cells (top number) and mean fluorescence intensity (bottom number) in the shRNA-expressing population. Empty vector does not contain shRNA; GFP shRNA targets the transcript encoding GFP and serves as a positive control for GFP knockdown. (c) Flow cytometry of GFP expression in ER-GADD45a-expressing cells treated for 18 h with tamoxifen with or without a p38 or Jnk inhibitor, analyzed and gated as described in a. (d) Immunoblot of total and phosphorylated (p-) p38 and Jnk in ER-GADD45a-expressing cells treated for 6 h as described in c. In the Jnk blot, the lower band is Jnk1 (p46) and the upper band is Jnk2 and Jnk3 (p54). All data are representative of at least two independent experiments.