Fig 1: Clathrin promotes Prolactin-induced STAT5 activation in LrBM-engaged MECs. (A,B) Eph4 cells transfected with CHC-specific SMARTpool (A) or individual oligos (B) were seeded onto plastic and LrBM added to the differentiation medium as appropriate. After 24 hours, cells were stimulated with Prl (3 µg/ml) as indicated for 15 mins before lysis. Samples were analysed by SDS-PAGE/western blotting with phospho-Y694 STAT5, total STAT5a or tubulin specific antibodies, and quantification of Odyssey scanned fluorescent images performed using ImageJ. (C) Eph4 cells transfected with Caveolin-1-specific SMARTpool or individual oligos were treated and analysed as in (A). Western blots are representative of, and graphs show normalised data from, at least 3 independent experiments. *p.0.05, **p < 0.01, ns = not significant.
Fig 2: Prolactin is internalised via CME in LrBM-engaged MECs. (A) Schematic of Prl internalisation assay. (B) Eph4 cells seeded onto plastic were overlaid with LrBM as appropriate and labelled with Prl (3 µg/ml) at 4 °C for 15 mins. Cells were either washed with PBS to reveal total bound Prl (Total), or acid washed to reveal internalised Prl (Internal), and samples analysed by SDS-PAGE and western blotting with Prl, pSTAT5, STAT5 and tubulin specific antibodies. (C) Eph4 cells were labelled with Prl (3 µg/ml) for 15 mins at 37 °C or 4 °C as indicated, and total and internal pools analysed as in (B). Quantification of Odyssey scanned fluorescent images performed using ImageJ. Yellow line indicates background signal in the absence of exogenous Prl. (D,E) Prl internalisation was analysed in CHC and Caveolin-1 knockdown cells (2 oligos each), background signal in the absence of exogenous Prl was subtracted to give relative internal Prl above background. Western blots are representative of, and graphs show normalised data from, at least 3 independent experiments. *p < 0.01, **p.0.001, ns = not significant.
Fig 3: Immunohistochemistry of ACP4, LAMP1, CLTC, and RAB5 Expression in Consecutive, Longitudinally-Sectioned D10 Mouse Mandibular Incisors. Sections were immunostained red using antibodies against mACP4 (A), hACP4 (B), CLTC (clathrin-coated vesicles; C), and RAB5 (early endosomes; D) or immunostained green for LAMP1 (lysosomes and late endosomes). DAPI was used to stain cell nuclei blue. a. Tri-stained (left) and individual antibody-stained (right) apical half of mandibular incisors with box outlines of regions shown in higher magnification in b and c); b. Early and c. Mid-secretory stage ameloblasts. d. Three higher magnification images of the distal region of secretory stage ameloblasts (outlined in b or c) and immunostained for the target protein (red, left), LAMP1 (green, middle); or tri-stained (right). Am, ameloblasts; Od, odontoblasts; TP, Tomes' processes; TW, terminal web. ACP4 and LAMP1 signals were minimally overlapping suggesting ACP4 is not a lysosomal enzyme. ACP4 localizes to the Tomes' processes distally (potentially in endosomes and/or the distal membrane) as well as in the ameloblast lateral plasma membrane. These results are consistent with ACP4 dephosphorylating enamel matrix proteins in the matrix near the plasma membrane and in early endosomes. More immunostaining is shown in Supplemental Figs. S10–S14.
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