Jansen EJ , Hafmans TG , Martens GJ .

	Figure 1. Generation of transgenic Xenopus laevis with expression of GFP-Ac45 specifically in the intermediate pituitary melanotrope cells. (A) Schematic representation of the linear transgene construct used for Xenopus transgenesis (pPOMC(A)2+-SP-GFP-Ac45) with the Xenopus POMC-A gene promoter fragment (pPOMC), Ac45 signal peptide (SP), the region encoding the GFP-Ac45 fusion protein, and the SV40 polyadenylation signal (pA). TM: transmembrane region. (B) Ventral view on the pituitary of a wild-type (wt) and GFP-Ac45 (#533) transgenic (tg) juvenile frogs. Fluorescence was detected in the intermediate lobe (IL) but not in the anterior lobe (AL) and the brain. (C) Sagittal cryosections of brain and pituitary from wild-type (wt) and Ac45-transgenic (tg) Xenopus. Transgene expression was detected by direct fluorescence and endogenous POMC expression by immunostaining with an anti-POMC antibody. NL, neural lobe.
	Figure 2. Excess Ac45 causes enhanced intragranular acidification in transgenic melanotrope cells. (A) Immunogold labeling of ultrathin sections of DAMP-incubated wild-type (wt) or Ac45-transgenic (tg) Xenopus melanotrope cells with the anti-DNP antibody in combination with 10-nm protein-A gold. Gold labeling was predominantly found in the secretory granules (arrows) and lysosomes (L). Bars equal 200 nm. (B) Quantification of intragranular DAMP accumulation. The number of gold particles per μm2 found in 205 wt and 206 tg dense-core secretory granules of 20 randomly selected wt or tg melanotrope cells from three wt or tg frogs.
	Figure 3. Proprotein processing in Ac45-transgenic melanotrope cells is less effectively inhibited by bafilomycin A1. (A and B) Neurointermediate lobes from wild-type (wt) and Ac45-transgenic (tg) animals were pulse labeled for 30 min and subsequently chased for 180 min in medium containing 0, 0.25, or 0.5 μM bafilomycine A1. Newly synthesized proteins were extracted from the lobes, directly analyzed on 15% SDS-PAGE to resolve the 37-kDa POMC and 18-kDa POMC products (A) and on 10% SDS-PAGE to resolve the various PC2 forms (B). Signals were visualized by autoradiography.
	Figure 4. Steady-state PC2 protein expression levels are lower in Ac45-transgenic melanotrope cells. (A) Western blot analysis of total proteins extracted from wild-type and Ac45-transgenic neurointermediates lobes of black-adapted Xenopus using various antibodies. (B–C) Steady-state protein levels of POMC (B), and PC2 (C) are presented in arbitrary units (AU) relative to the steady-state levels of calnexin.
	Figure 5. Excess Ac45 affects the processing of newly synthesized POMC in transgenic melanotrope cells. (A) Neurointermediate lobes (NILs) from wild-type (wt) and Ac45-transgenic (tg) animals were pulse labeled for 30 min and subsequently chased for the indicated time periods. 5% of the newly synthesized proteins extracted from the lobes and 20% of the proteins secreted into the incubation medium were directly resolved by 12.5% SDS-PAGE and visualized by autoradiography. The experiments were performed in triplicate, and a representative example is shown. (B) The processing rate of 37-kDa POMC to 18-kDa POMC and the secretion of 18-kDa POMC were quantified by densitometric scanning using a phosphoimager. For each chase period, the ratio of 18-kDa POMC to 37-kDa POMC was calculated for wild-type (wt, white bars) and transgenic (tg, black bars) NILs. The amount of secreted 18-kDa POMC was calculated relative to the levels of newly synthesized actin and is presented in AU. Note that the newly synthesized Ac45-transgene product comigrated with mature PC2 (Jansen et al., 2008). Shown are the means ± SEM (n = 3). Significant differences are indicated by *(p < 0.05) or **(p < 0.01).
	Figure 6. ProPC2 maturation and pro7B2 processing are delayed in Ac45-transgenic melanotrope cells. (A) Wild-type (wt) and Ac45-transgenic (tg) neurointermediate lobes (NILs) were pulse labeled for 20 min and subsequently chased for indicated time periods. After protein extraction, immunoprecipitation analyses were performed using an anti-PC2 antibody. (B) Quantification of PC2 maturation was calculated from the matPC2: proPC2 ratio. (C) Pulse-chase-labeled NIL proteins were immunoprecipitated using an anti-7B2 antibody. (D) Quantification of 7B2 processing was calculated from the 18-kDa 7B2:25-kDa 7B2 ratio. Shown are the means ± SEM (n = 4). Significant differences are indicated by *(p < 0.05). (E) Pulse-chase-labeled NIL proteins were extracted under native conditions, and proPC2 and 7B2 were coimmunoprecipitated under native conditions using an anti-PC2 antibody; the PC2 gel was exposed for one day and the 7B2 gel for 12 d. (F) Quantification of the amount of 25-kDa 7B2 binding to proPC2 was calculated from the 25-kDa 7B2: proPC2 ratio; the difference was not statistically significant (p = 0.3).
	Figure 7. Ac45-transgenic melanotrope cells secrete less α-MSH than wild-type cells. (A) Wild-type (wt) and Ac45-transgenic (tg) neurointermediate lobes were isolated and directly superfused. To measure the amount of α-MSH released into the medium, fractions of the superfusion medium were collected and subjected to an α-MSH radioimmunoassay. The average α-MSH release during a ∼50-min period (7 fractions) was measured. The amount of α-MSH released was normalized for calnexin levels (see B) and the release of α-MSH by wild-type cells was set to 100%. Shown are the means ± SEM (n = 4). Significant difference is indicated by *(p < 0.05). (B) To estimate the number of wt and tg melanotrope cells, after superfusion the amount of the reference protein calnexin was determined by Western blot analysis.
	Figure 8. Processing of POMC by PC1 and PC2. Simplified version of the POMC processing scheme of Bicknell (Bicknell, 2008). Only the cleavage events pertinent to the present study are indicated. Filled dot, N-linked glycosylation site; MSH, melanophore-stimulating hormone; JP, joining peptide; CLIP, corticotropin-like intermediate lobe peptide.

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