Fig. 2.

LDAH is absent in mice with a targeted gene-disruption allele. A: A gene knockout cassette disrupts exons 2 and 3 of the Ldah gene. B–E: Ldah mRNA and protein are absent in Ldah KO animals. Schematic of the Ldah gene locus and targeting cassette (Knockout Mouse Project). Genotype of animals was confirmed with SD30636, NeoF, and SD primers. B: Ldah mRNA is reduced to 50% of the gene product in heterozygous mice and absent in Ldah KO animals. Relative Ldah mRNA abundance ± SD in different tissues of Ldah WT (black bars), heterozygous (gray bars), and KO mice (white bars) determined by qPCR. Ldah values were normalized to the average of β-actin and cyclophilin. Values are means (n = 3–4). C: Western blots against LDAH in liver tissue from male and female Ldah WT and KO animals confirmed loss of LDAH protein. Tubulin was used as a loading control. D: LDAH protein is undetectable in tissues of male Ldah KO animals by Western blot. Low and high exposures are shown for tubulin. E: Mass spectrometry (MS) analysis confirmed absence of LDAH in Ldah KO animals. Peptides that were identified by MS for LDAH (top) or ATGL (bottom) in WT or Ldah KO animals are mapped to the protein sequence. For ATGL, peptides were identified in both WT and KO tissue across the length of the protein. For LDAH, no peptides were identified in KO animals, and peptides from various parts of the protein were detected in lysates from WT tissue. Data from WAT and livers of two animals per genotype were combined for the graph.