Figure 3

Liver-specific deletion of STAT3. (a) Full-length STAT3 protein is not detectable in liver of Cre⁺; Stat3^flox/D mice: indicated tissues from Stat3^+/+, Cre⁺; Stat3^flox/D and Stat3^flox/D mice were homogenized and the supernatant was separated by 8% SDS-PAGE and immunoblotted with anti-STAT3 (H190, Santa Cruz) antibody. The gene product of Cre⁺; Stat3^flox/D mutant was a partial deletion of Stat3 gene (exons 18, 19 and 20). A major band at 90 kDa representing STAT3 is completely and specifically missing in the liver of Cre⁺; Stat3^flox/D mice. (b) STAT3 mRNA is truncated in hepatocytes carrying Stat3 flox or Stat3 D alleles: RT-PCR using RNA isolated from hepatocytes from Stat3^+/+, Cre⁺; Stat3^flox/D and Stat3^flox/D mice was performed. After reverse transcription cDNA was amplified by PCR using a primer pair binding to exons 13 and 21, respectively. The shorter PCR product (530 bp) shows the presence of a truncated form of STAT3 mRNA in Stat3^flox/D mice indicating the specific deletion of the targeted gene fragment. (c) STAT3 binding activity is abolished in hepatocytes from liver-specific Stat3 KO mice: nuclear extracts from Stat3^+/+ and Cre⁺; Stat3^flox/D mice stimulated with or without 100 ng/ml hEGF or OSM were incubated with radiolabeled STAT3 binding site (m67-SIE) and were analyzed by EMSA. No STAT3 DNA binding was found in Cre⁺; Stat3^flox/D mice. (d) Body weight of the Cre⁺; Stat3^flox/D (L-Stat3^−/−) and Stat3^flox/+ mice. The L-Stat3^−/− mice were not obese.