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Fig. 5
Plin6 is localized to carotenoid droplets and is phosphorylated under conditions that disperse pigment.
(A) Western blot analysis of Plin6 expression from three fractions of skin homogenate separated by a sucrose gradient; total protein homogenate in 40% sucrose prior to centrifugation (Input), the protein fraction at the interface of the 20% and 40% sucrose layers following centrifugation (20/40), and the purified CD fraction that floated above the 20% sucrose following centrifugation (CD). (B) Western blot analysis of Plin6 expression from untreated skin (adult skin), and from scales treated with epinephrine (Epi) or forskolin-IBMX (FSK) to inhibit or stimulate PKA activity, respectively. Proteins from treated scales were resolved using polyacrylamide gels containing Phos-tag, which retards the electrophoretic mobility of phosphorylated proteins. Plin6 is largely unphosphorylated (Unphos, white arrowheads) under conditions that promote aggregation (Epi) and highly phosphorylated (Phos, grey arrowheads) under conditions that promote dispersion (FSK). (C) Plin6 immunoTEM in zebrafish scales. (C-1) A dotted white line demarks the rough boundary between a xanthophore (top) and melanophore (bottom). The carotenoid body (CB) with clusters of CD puncta is located in a field adjacent to the nucleus (Nu) of the xanthophore. Pterinosomes (p) within the xanthophore are also present, as well as a field of melanosomes (m) within the adjacent melanophore. (C-2) Higher magnification of the boxed inset shown in C-1. (C-3) Higher magnification of the boxed inset shown in C-2 show Plin6 immunoreactivity targeted to clusters of CDs (arrowhead).