FIG. 1.
Hzf is induced by p19Arf or IR. (A) NIH 3T3 and MT-Arf cells were treated with 80 μM zinc sulfate for the indicated periods, and Hzf and Mdm2 mRNAs were detected by Northern blotting. 28S and 18S rRNAs in the same membrane were stained with methylene blue before hybridization, confirming that equal amounts of RNA were loaded in each lane. (B) NIH 3T3 and MT-Arf cells treated for 24 h with zinc sulfate as indicated were stained with purified rabbit polyclonal antibodies directed to a C-terminal peptide of Hzf (left panels). Cells were counterstained with DAPI (4′,6′-diamidino-2-phenylindole) (right panels). (C) NIH 3T3 cells were exposed to 5 Gy of IR, and RNAs prepared at the indicated times were amplified by RT-PCR (top two panels). RT-PCR products were subjected to Southern blotting using Hzf cDNA as a probe (DNA blot, middle panel). Lysates were prepared from the same samples after IR, and Hzf protein was detected by immunoblotting using Cdk4 protein as a loading control (bottom two panels).