Fig 1.
Generation of CerS6ADAAAIA mice.
The CerS6 genomic region showing the CRISPR/Cas9 design for editing the CerS6 DDRSDIE motif. Genomic location (mm10 chr2:68,861,441–69,114,282) of the guide RNA cleavage site is shown as a dashed line. The single strand (ssDNA) repair oligo spanning 60 base pairs of the genomic sequence up- and down-stream of the edited motif is indicated. The genomic and repair sequences are shown, and amino acid residues that were modified are in red. Insert, mouse kidney homogenates (20 μg of protein) were analyzed by Western blotting using an anti-CerS6 antibody, repeated twice with similar results. Mr markers are shown. GAPDH was used as a loading control.
Fig 2.
In vitro ceramide synthase activity in tissues from the CerS6ADAAAIA mouse.
CerS activity was assayed using 20 μg of protein from homogenates from the indicated tissues, and C16:0-CoA was used as substrate. Data are means ± S.D., n = 4. No significant differences were detected.
Fig 3.
Dihydroceramide and ceramide levels in the small intestine of the CerS6ADAAAIA mouse.
Fatty acyl composition of d18:0- and d18:1-ceramides were measured by LC-MS/MS. Data are means ± S.E.M., n = 4. *, p<0.05; **, p<0.01; ***, p<0.001.
Fig 4.
Dihydroceramide and ceramide levels in the liver of CerS6ADAAAIA mouse.
Fatty acyl composition of d18:0- and d18:1-ceramides were measured by LC-MS/MS. Data are means ± S.E.M., n = 4. *, p<0.05; **, p<0.01; ***, p<0.001.