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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Nat Cell Biol. 2019 Apr 1;21(4):420–429. doi: 10.1038/s41556-019-0301-x

Figure 4. Light-dependent activation of phospho-T53-DAT/SLC6A3 in the IPL requires OPN5.

Figure 4.

a, b, Labelling of P8 Opn5+/+ retina for nuclei (blue) and phospho-T53-DAT (green) for dark adapted mice half of which were exposed to 30 minutes of 380 nm light. NBL: neuroblastic layer. IPL: inner plexiform layer. GCL: ganglion cell layer. NFL: nerve fibre layer. Scale bars 50 μm. Indicated areas of each panel are exported to chart (e). e, Chart showing averaged (n=3) quantification profiles (dark adapted, grey trace, light exposed, blue trace) for phospho-T53-DAT in retinal cryosections (a, b). Grey shading emphasizes the elevated signal of light-induced sample. c, d, As in (a, b) but for Opn5−/− retina. f, Chart as in (e) except for Opn5−/− mice (dark-adapted, orange trace, or dark adapted and exposed to 380 nm light for 30 minutes, red trace). Traces from chart (e) are reproduced on chart (f) for comparison. g, h, Charts showing under-the-peak quantification (n=3) of phospho-T53-DAT labelling in inner plexiform layer (g) and nerve fibre layer (h) for P8 mice of the labelled genotype and light exposure. The vertical dashed lines in (e, f) indicate the areas that were quantified. i, Immunoblot detecting DAT/SLC6A3 (DAT), phospho-T53-DAT/SLC6A3 (pDAT) and β-tubulin (TUBB) in retinae harvested from Opn5+/+ (left lane) and Opn5−/− (right lane) mice during the light phase. phospho-T53-DAT/SLC6A3 is lower than normal in the Opn5 null. Unprocessed blot image in Supplementary Figure 6. j, Quantification of P8 hyaloid vessels in WT mice injected with vehicle or with the DAT/SLC6A3 inhibitor raised from E17 either in normal (LD) lighting, or in constant darkness (DD). k, Quantification of P8 hyaloid vessels in Opn5+/+, Opn5+/−, and Opn5−/− mice injected from P1-P8 with the DAT/SLC6A3 inhibitor. p-values by Two Way ANOVA. Error bars are SEM. The number at the base of each chart is n and represents the number of animals assessed. Panels a-d are representative of 3 separate experiments.