FIG. 5.

Klf3 expression during 3T3-L1 adipocyte differentiation. (A) Total RNA was extracted from 3T3-L1 cells at the indicated times before and after induction of differentiation. The expression levels of Klf3, Klf2, Klf15, and C/ebpα were determined by quantitative real-time RT-PCR and normalized to 18S rRNA levels. These levels were then further normalized with respect to the level in 3T3-L1 cells at day 0, set at 1. Results are expressed as means plus SEM (n = 3). (B and C) Nuclear extracts were made from cells at day 0 (preadipocytes) and day 10 (adipocytes) of 3T3-L1 differentiation. An EMSA using a double-stranded oligonucleotide probe containing a CACCC sequence shows that Klf3 is present in preadipocytes and reduced in adipocytes (B). Anti-Klf3 antibody supershift reactions confirm that these species are Klf3 (lanes 2 and 4). Note that supershift of the Klf3 band reveals the presence of another CACCC box binding protein that runs similarly to Klf3. All data are representative of at least three independent experiments. Klf3 protein was detected by Western blot analysis using an anti-Klf3 antibody (C). β-Actin was used as a loading control. Duplicates are shown for each condition. All data are representative of at least three independent experiments.