Confirmation of hepatocyte Smad1 and/or Smad5 ablation in conditional knockout mice. (A) Schematic depictions of loxP-flanked (floxed) Smad1 or Smad5 allele and the allele after Cre recombinase-mediated excision. F and R indicate forward and reverse primers used for PCR genotyping (left). PCR analysis of genomic DNA extracted from total liver (containing both hepatocytes and nonparenchymal cells) of double-knockout Smad1^fl/fl;Smad5^fl/fl;Cre⁺ mice and littermate Cre⁻ controls at 8 weeks of age (right). (B-C) Relative Smad5 (B) and Smad1 (C) mRNA levels in the total liver (n = 7-9 per group; 8 weeks of age) and isolated hepatocytes (n = 3-4 per group; 6 weeks of age) of Smad5^fl/fl;Cre⁺, Smad1^fl/fl;Cre⁺, and Smad1^fl/fl;Smad5^fl/fl;Cre⁺ mice compared with their respective littermate Cre⁻ controls. Transcripts were normalized to Rpl19, and the average of the respective Cre⁻ control mice was set to 1. Values represent mean ± standard error of the mean. ***P < .001 relative to the respective Cre⁻ controls by Student t test. (D) Western blot analysis of Smad5 and Smad1 in the livers of Smad5^fl/fl;Cre⁺, Smad1^fl/fl;Cre⁺, and Smad1^fl/fl;Smad5^fl/fl;Cre⁺ mice compared with their respective littermate Cre⁻ controls at 8 weeks of age. Actin is used as a loading control.