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Fig. 1.
Analysis of the p24 system in the transgenic Xenopus intermediate pituitary melanotrope cells: (A) the amino acid sequences of the C-terminal tails of the wild-type and mutated p24δ2 transgene products with the mutated amino acids indicated in bold. (B) Schematic depiction of the linear injection fragments pPOMC-p24δ2-GFP (δ2 Bouw et al., 2004 and Strating et al., 2007), pPOMC-p24δ2 FF/AA-GFP (FF), and pPOMC-p24δ2 KK/SS-GFP (KK), containing a Xenopus POMC gene promoter fragment (pPOMC), which drives transgene expression specifically to the melanotrope cells ( Jansen et al., 2002), and the protein-coding sequence of the wild-type (wt) p24δ2-GFP, p24δ2 FF/AA-GFP, and p24δ2 KK/SS-GFP, respectively. (C) Western blot analysis of neurointermediate lobe (NIL) lysates from nontransgenic (nt) frogs and frogs transgenic for p24δ2 FF/AA (FF; line #226) or p24δ2 KK/SS (KK; line #241) with an anti-GFP antibody. Tubulin was used as a control for equal loading. (D) Immunoprecipitation analysis of lysates from [35S]-methionine/cysteine-labelled NILs of nt frogs and frogs transgenic for wt p24δ2 (δ2; line #124 Bouw et al., 2004), p24δ2 FF/AA (line #226) or p24δ2 KK/SS (line #241). An anti-GFP antibody was used. (E) Western blot analysis of NIL lysates from nt frogs and frogs transgenic for p24δ2 FF/AA (FF; line #226) or p24δ2 KK/SS (KK; line #241). Anti-p24 antibodies were used and tubulin served as a control for equal loading. (F) NIL lysates were control treated (−) or deglycosylated with peptidyl N-glycosidase F (PNGaseF; F; removes all N-linked glycans irrespective of their conformation) or endoglycosidase H (EndoH; H; removes only high-mannose but not complex N-glycans) and analysed by Western blotting with an anti-p24α3 antibody. EndoH-sensitive p24α3 (asterisk) was only observed in samples from the p24δ2 FF/AA-transgenic cells. Since the band corresponding to EndoH-sensitive p24α3 was faint, we have digitally increased the contrast of the blot (lower panel). (G) Confocal laser scanning microscopy analysis of live melanotrope cells with transgene expression of wt p24δ2-GFP (line #124), p24δ2 FF/AA-GFP (line #226) or p24δ2 KK/SS-GFP (line #241), showing GFP-fluorescence in Golgi-like perinuclear structures (δ2) or reticular structures (possibly ER) throughout the cells (FF and KK). The strengths of the GFP-signals do not reflect the actual transgene-expression levels, since for the various panels the exposure times differed. (H) Immuno-electron microscopy analysis of melanotrope cells from nt frogs (H1) and frogs transgenic for p24δ2 FF/AA (line #226; H2) or p24δ2 KK/SS (line #241; H3 and H4). An anti-GFP antibody was used. In the nt cells only a few, non-specific gold particles were found dispersed throughout the cell. In the p24δ2 FF/AA-tmcs the gold particles were largely found on ER membranes. In the p24δ2 KK/SS-tmcs, the staining was mainly found in the Golgi/immature secretory granule compartment, whereas the ER contained only a few gold particles. Abbreviations used: er, rough endoplasmic reticulum; g, Golgi; l, lysosome; m, mitochondrion; n, nucleus; s, immature secretory granule. Bars equal 5 μm (G), 500 nm (H1–H4).
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Fig. 2.
Steady-state levels of secretory cargo proteins in wild-type and transgenic Xenopus intermediate pituitary cells, Western blot analysis of neurointermediate lobe (NIL) lysates from nontransgenic (nt) frogs and frogs transgenic for wt p24δ2 (δ2; line #124 Bouw et al., 2004), p24δ2 FF/AA (FF; line #226) or p24δ2 KK/SS (KK; line #241) using antibodies directed against the soluble secretory cargo proteins proopiomelanocortin (POMC) and prohormone convertase 2 (PC2), and the transmembrane cargo amyloid-β precursor protein (APP). Tubulin was used as a control for equal loading.
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Fig. 3.
The effect of p24δ2 FF/AA- or p24δ2 KK/SS-transgene expression on POMC biosynthesis and processing in Xenopus melanotropes: (A) neurointermediate lobes (NILs) from nontransgenic (nt) frogs and frogs transgenic for wt p24δ2 (δ2; line #124) (Bouw et al., 2004), p24δ2 FF/AA (FF; line #226) or p24δ2 KK/SS (KK; line #241) were pulse labelled with [35S]-Met/Cys for 30 min. Newly synthesised proteins extracted from the NILs were resolved by 15% SDS-PAGE and visualised by autoradiography. (B) The amount of newly synthesised 37K POMC produced during the 30 min pulse period (panel A) in nt (n = 4) and the wt p24δ2-transgenic (n = 3), p24δ2 FF/AA-transgenic (n = 3) and p24δ2 KK/SS-transgenic (n = 3) NILs was quantified and is shown relative to the nt cells. (C) NILs were pulsed for 30 min and subsequently chased for 180 min. Newly synthesised proteins extracted from the NILs (Cells) or secreted into the incubation medium (Media) were resolved by 15% SDS-PAGE and visualised by autoradiography. (D) The amount of newly synthesised 37K POMC remaining in the cells after the chase period (panel C) in nt (n = 6) and the wt p24δ2-transgenic (n = 7), p24δ2 FF/AA-transgenic (n = 6) and p24δ2 KK/SS-transgenic (n = 6) cells was quantified and is shown relative to the nt cells. (E) The total amounts (cells + media) of newly synthesised ∼18K POMC processing products (18K + 18K* POMC) after the chase period (panel C) in nt (n = 6) and the wt p24δ2-transgenic (n = 3), p24δ2 FF/AA-transgenic (n = 6) and p24δ2 KK/SS-transgenic (n = 6) cells were quantified and are shown relative to nt. (F) The total amounts (cells + media) of newly synthesised 18K and 18K* POMC processing products after the chase period (panel C) in nt (n = 6) and the wt p24δ2-transgenic (n = 4), p24δ2 FF/AA-transgenic (n = 6) and p24δ2 KK/SS-transgenic (n = 6) cells were quantified and are shown relative to wt 18K POMC. Indicated are the 18K/18K* ratios and their statistical evaluations. Data are shown as means ± S.E.M. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
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Fig. 4.
Sulphation of newly synthesised POMC in wild-type and transgenic Xenopus intermediate pituitary cells, Neurointermediate lobes (NILs) from nontransgenic (nt) frogs and frogs transgenic for wt p24δ2 (δ2; line #124 Bouw et al., 2004), p24δ2 FF/AA (FF; line #226) or p24δ2 KK/SS (KK; line #241) were pulse labelled with 35S-sulphate and 3H-lysine for 15 min and the amount of each label incorporated into newly synthesised 37K POMC was determined. Shown are the amounts of newly synthesised sulphated 37K POMC produced in the transgenic relative to nt NILs. Data are shown as means ± S.E.M. (wt, n = 12; transgenics, n = 3); n.s., not significant.
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Fig. 5.
Electron microscopy analysis of wild-type and transgenic melanotrope cells: (A–H) Ultrastructural electron microscopy analysis of glutaraldehyde-fixed and Epon-embedded neurointermediate lobes (NILs) from nontransgenic (nt) frogs (A and B) and frogs transgenic for wt p24δ2 (δ2; line #124 Bouw et al., 2004) (C and D), p24δ2 FF/AA (FF; line #226) (E and F) or p24δ2 KK/SS (KK; line #241) (G and H). In the left column (A, C, E and G) the tissues are displayed at low magnification, showing a similar overall tissue organisation between the nt and the wt p24δ2-, p24δ2 FF/AA- and p24δ2 KK/SS-transgenic NILs with clearly visible nuclei and well-developed rough endoplasmic reticulum. At a higher magnification as displayed in the right column (B, D, F and H), various subcellular structures such as the nucleus, the ER, the Golgi apparatus, mitochondria and dense-core immature secretory granules can be readily identified in the nt melanotropes and in the wt p24δ2-, p24δ2 FF/AA- and p24δ2 KK/SS-tmcs. Dotted lines highlight the outline of the Golgi. Overall the structures of the transgenic cells were similar to those of the nt cells, except that the Golgi could be more readily identified, appeared to be more extensively present and Golgi ribbons seemed to be longer in the p24δ2 KK/SS-tmcs, which may suggest an expansion of the Golgi or more extensive lateral linking individual stacks: er, rough endoplasmic reticulum; g, Golgi; l, lysosome; m, mitochondrion; n, nucleus; pm, plasma membrane; arrowheads, dense-core immature secretory granules. Scale bars equal 5 μm (A, C, E and G); 1 μm (B, D, F and H).
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Figure S1. Generation of Xenopus laevis with transgene expression of p242 FF/AA or p242 KK/SS specifically in the intermediate pituitary melanotrope cells
(A) Pituitary-specific GFP-fluorescence (arrows) in living tadpoles transgenic for p242 FF/AA-GFP (FF; line #226) or p242 KK/SS-GFP (KK; line #241); G, gut; E, eye; N, nose. (B) Sagittal cryosections through the brain and pituitary of adult frogs transgenic for p242 FF/AA-GFP (FF; line #226) or p242 KK/SS-GFP (KK; line #241) show GFP-fluorescence only in the IL but not in the AL of the pituitary. POMC is detected with a primary antibody recognising only the 37K prohormone and a Texas red-conjugated secondary antibody. The bars equal 100m.
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Figure S2. Western blot analysis of endogenous p241 and p242 in melanotropes from different lines with transgene expression of p242 FF/AA or p242 KK/SS
Western blot analysis of NIL lysates from frogs transgenic for p242 FF/AA (FF; lines #235 and #226) or p242 KK/SS (KK; lines #247, #248 and #241) with an antibody against the C-terminus of p241 and p242.
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Figure S3. POMC biosynthesis in Xenopus melanotropes of various transgenic lines with transgene expression of p242 FF/AA or p242 KK/SS
(A) Neurointermediate lobes (NILs) from frogs transgenic for p242 FF/AA (FF; lines #235 and #226) or p242 KK/SS (KK; lines #247, #248 and #241) were pulse labelled with [35S]-Met/Cys for 30 min. Newly synthesised proteins extracted from the NILs were resolved by 15% SDS-PAGE and visualised by autoradiography. (C) NILs were pulsed for 30 min and subsequently chased for 180 min. Newly synthesised proteins extracted from the NILs were resolved by 15% SDS-PAGE and visualised by autoradiography.
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Figure S4. Electron microscopy analysis of p242 FF/AA- and p242 KK/SS-transgenic melanotrope cells.
(A-D) Ultrastructural electron microscopy analysis of intermediate pituitary cells from p242 FF/AA- [lines #235 (A) and #226 (B)] and p242 KK/SS-transgenics frogs [lines #247 (C) and #241 (D)] did not reveal gross differences between the different lines transgenic for the same construct. er, rough endoplasmic reticulum; g, Golgi; ics, intercellular space; l, lysosome; m, mitochondrion; n, nucleus; pm, plasma membrane; arrowheads, dense-core immature secretory granules. Scale bars equal 2μm (A-B); 1μm (C- D).
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