FIG. 2.

Time course of RA-regulated expression of RARβ₂ and RARγ₁. (A) SK-N-BE2(c) cells were plated at 10⁶ cells per 25-cm² tissue culture flask, and after an overnight incubation at 37°C, RA was added (time zero) to a final concentration of 10 nM. At various times after RA addition, total RNA was isolated and 1 μg was analyzed by RT-PCR for RAR or RXR expression as described in the text. Values for RAR and RXR mRNAs were normalized to that for β-actin mRNA used as internal standard for each RNA sample. The degree of amplification was quantitated by scanning densitometry and plotted as a ratio of RAR to β-actin or RXR to β-actin. Only data relative to RARβ₂ and RARγ₁ are reported, since no modulations were observed for the remaining RARs and RXRs. Five independent experiments with very similar results were conducted. OD, optical density. (B) Ten micrograms of DNA-binding proteins obtained from control cells and cells exposed to 10 nM RA was electrophoresed on SDS-polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies against RARα, RARγ, or RXR. Lanes 1, control cells; lanes 2, cells exposed to RA for 90 min; lanes 3 to lane 9, treated cells collected every 30 min. Prestained molecular size standards were used to identify bands of the correct molecular weight.