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. 2017 Feb 22;292(14):5884–5897. doi: 10.1074/jbc.M117.776914

FIGURE 7.

FIGURE 7.

Antagonistically bifunctional activity of RDH10-DHRS3 complex provides independence from fluctuations in the levels of individual components. A, Western blotting and activity analysis of HEK 293 cells co-transfected with a fixed amount of RDH10-FLAG construct and increasing amounts of DHRS3-FLAG construct. The ratio of DHRS3/RDH10 plasmid varied from 0 to 2 (μg/μg), as indicated. RA production was determined by HPLC after incubating the cells with 2 μm retinol for 9 h (means ± S.D., n = 3). B, HEK 293 cells were transfected with increasing amounts of RDH10-expressing construct (1–3 μg as indicated) separately or together with a fixed amount of bicistronic vector (RDH10-IRES-DHRS3). RA production was measured as in A. Note that RA production levels in the cells co-transfected with RDH10 and bicistronic vector remain stable despite the gradual increase in RDH10 protein. C, Western blotting and activity analysis of HEK 293 cells co-transfected with a fixed amount of RDH10-FLAG and increasing amounts of Y188A DHRS3-FLAG. Note the gradual increase in RA production, reflecting the loss of homeostasis. IB, immunoblot.