5-Aza-CdR treatment activates STATs 1, 2, and 3 in HT29 cells.
(A) The expression level of IFN-α in HT29 cells before
and after 500 nM 5-Aza-CdR treatment was measured by reverse
transcription–PCR along with gapdh to confirm equivalent cDNA input.
(B) STAT transcription factor levels were measured by
Western blotting. Cytoplasmic (C) and nuclear (N) cell extracts were
prepared from HT29 cells after treatment with vehicle or 500 nM
5-Aza-CdR. A poly(vinylidene difluoride) membrane harboring the protein
extracts was probed sequentially with mAbs specific to STATs 1, 2, and
3. In each case, the antibodies recognized proteins of the appropriate
molecular weight for each STAT. Molecular mass markers are indicated.
(C) The expression of STAT 1, 2, and 3 genes was
measured by Northern blotting. RNA was isolated from HT29 cells after
treatment with vehicle or 500 nM 5-Aza-CdR. The locations of molecular
mass markers are indicated. Ethidium bromide staining confirmed equal
RNA loading (28S, 18S rRNAs).