Figure 2.

Protein levels of RDH10 and DHRS3 in hepatic membrane fractions from fed versus fasted mice.A–C, side-by-side western blot analysis of hepatic subcellular fractions from mice fasted for 16 h. Subcellular fractions were isolated from four individual 3-month-old male mice and loaded as follows: 40 μg of hepatic microsomes (A); 40 μg of mitochondria (B); and lipid droplets loaded by wet weight (10 μl out of 200 μl) (C). The blot was incubated with a mixture of RDH10 and DHRS3 antibodies diluted 1:3000 each. After developing the blots, they were incubated with the corresponding markers of each subcellular fraction: P450 reductase (P450red), antibodies diluted 1:4000; pyruvate dehydrogenase complex subunits E2 and E1α, antibodies diluted 1:5000; and ADRP (perilipin 2), antibodies diluted 1:3000. D, western blot analysis of RDH10 and DHRS3 protein content in mitochondria (MT), microsomes (MS), and lipid droplets (LD) isolated from 3-month-old male mice (n = 3) fed or fasted for 24 h. Each membrane fraction was resuspended in 200 μl, the MS and MT membrane fractions (but not LD fraction) were diluted fivefold and 10 μl of each fraction was loaded onto two separate gels. The last sample set represents the average of fractions from individual mice mixed together (FED av and FASTED av). A representative Ponceau-stained blots showing protein amount in each lane are depicted in Fig. S5A. E, quantification of RDH10 and DHRS3 proteins in hepatic membrane fractions from fed and 24 h-fasted mice. The blots were scanned using UN-SCAN-IT and the data are presented as total pixel density. ∗p < 0.05; ∗∗p < 0.01. Error bars, S.D. Abbreviations are: LD_hf, lipid droplet fraction from the livers of mice on high-fat diet; RP, recombinant untagged human RDH10 and DHRS3 proteins overexpressed in HEK293 cells. Positions of molecular mass standards are indicated by numbers; asterisks (∗) denote a nonspecific protein band recognized by RDH10 antibodies in mitochondrial membranes.