Stürmer R , Reising J , Hoffmann W .

	Figure 1. Schematic structure of a mucous gland from X. laevis skin (A) as well as the integumentary mucins FIM-A.1 and FIM-C.1 (B). (A) The postulated migration of ordinary mucous cells towards the base of the gland during self-renewal is indicated by arrows. Also shown are the different types of secretory granules in ordinary mucous and cone cells, respectively. (B) The TFF domains in FIMs are shown in green, highly O-glycosylated regions typical of mucins are indicated by hexagons, and a potential N-glycosylation site is indicated with a square. The arrow in FIM-A.1 represents the cleavage site in the precursor by signal peptidase.
	Figure 2. FPLC purification and analysis of FIM-A.1 and FIM-C.1 from a ventral X. laevis skin extract. (A) Elution profile after SEC on an S-500 column as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). (B) Distribution of the relative FIM-A.1 (green) and FIM-C.1 content (black) as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the characteristic ≥ 116,000 band (antiserum anti-FIMA-1) or the ≫ 116,000 smear (antiserum anti-FIMC-1). The mucin content was semi-quantitatively analyzed using the PAS reaction (pink; Complexes I and II are marked). (C) 15% SDS-PAGE under reducing (R) or non-reducing (NR) conditions and subsequent Western blot analysis of the fractions B5, D3, and B9 using anti-FIMA-1. Molecular mass standard: left. (D) 15% SDS-PAGE under reducing conditions of the fractions B5, B9, D2, and D7 and Western blot analysis using antiserum anti-FIMA-1 or anti-FIMA-2. (E) 1% AgGE of the fractions B6, C12, D5, and D8 and Western blot analysis using anti-FIMA-1 or anti-FIMB-1. Molecular mass standard (M): FCGBP from human colonic mucus. (F) 15% SDS-PAGE of fractions B2–B10 under reducing conditions and Western blot analysis using anti-FIMC-1. (G) 15% SDS-PAGE under reducing conditions of fractions D1–D9 and Western blot analysis using anti-FIMB-1.
	Figure 3. Analysis of X. laevis FIM-A.1 by SEC after reduction. Fractions D6 + D7 (1.5 mL each) from SEC of Figure 2 were reduced in boiling 4.7% β-mercaptoethanol/0.1% SDS and separated immediately thereafter by SEC on an S-500 column. (A) Elution profile as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). Relative FIM-A.1 content (green), as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the characteristic ≥ 116,000 band (anti-FIMA-1). (B) 15% SDS-PAGE under non-reducing conditions and Western blot analysis of fractions D1–E3 using anti-FIMA-1. (C) 1% AgGE of fractions D1–D9 and Western blot analysis using anti-FIMA-1; for comparison (C): fraction D7 from Figure 2 (S-500 before reduction).
	Figure 4. Purification of X. laevis FIM-A.1 by SEC and anion-exchange chromatography. (A) Fractions D4 + D5 (1 mL each) from the SEC of Figure 2 were separated by anion-exchange chromatography on Resource Q6. Elution profile as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). (B) 15% SDS-PAGE of fractions B9–C3 under reducing conditions and Western blot analysis using anti-FIMA-1. (C) 15% SDS-PAGE of fractions B9–C2 under non-reducing conditions and silver staining. For comparison, Western blot analysis of fraction C1 using anti-FIMA-1. (D) Separation of fractions D8 + D9 analogous as described in (A). (E) Western blot analysis of fractions B10–C4 using anti-FIMA-1. (F) Silver staining of fractions B10–C1 and Western blot analysis of fraction C1 using anti-FIMA-1.
	Figure 5. In vitro binding of 125I-labeled FIM-A.1 with mucin fractions from X. laevis skin (overlay assay). FIM-A.1 was purified from X. laevis skin via SEC (from Figure 2) followed by anion-exchange chromatography of fractions D4 + D5 (see Figure 4A). Then, fraction B11 containing purified FIM-A.1 (Figure 4A) was labeled with 125I. 1% AgGE of fractions B1–C10 after SEC from Figure 2 and hybridization of the blot with 125I-FIM-A.1 (autoradiography). The start and the dye bromophenol blue (BPB) are marked on the left. For comparison, FIM-A.1 was detected on the same blot by immunostaining with anti-FIMA-1 (WB, Western blot; C9).

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