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. 2007 Aug 2;104(33):13461–13466. doi: 10.1073/pnas.0702239104

Fig. 2.

Fig. 2.

AgAPN1 expression and recognition by α-AgAPN1 PAbs. (a) RT-PCR analysis of AgAPN1 expression in guts from sugar-fed (0 h), blood-fed (24 h and 36 h PBF), and infected blood-fed (I-PBF) A. gambiae. Mosquito ribosomal S7 gene (AgS7) was used as a loading control. (b) Immunoblot of midgut lysates prepared at the indicated time points after a noninfected bloodmeal. Antibody to actin was used as a loading control. The upper arrowhead indicates the expected size of the predicted full-length AgAPN1 product. Preimmune serum does not recognize mosquito midgut antigens (SI Fig. 4). The lower arrowhead indicates a presumed degradation product of AgAPN1. (c) Immunofluorescence analysis of midgut cross-sections from A. gambiae fed on sugar (A and C) or on P. falciparum-infected blood (B and D) and stained with α-AgAPN1 PAb. Bright-field images of midguts from sugar-fed (A) and infected blood-fed (24 h PBF) (B) mosquito. C shows a fluorescence image of the same field as in A and shows α-AgAPN1 PAb staining of the microvilli (MV; arrow/bracket) as detected by FITC-labeled secondary antibody (green) with Evans blue as a counterstain (red). D is a fluorescence image of the same field as in B. The ookinete was stained with anti-Pfs25 antibody (green). α-AgAPN1 PAb stains the midgut MV–bloodmeal interface (arrowhead red). Note that AgAPN1 is present on the MMV during ookinete invasion. EC, epithelial cell; BM, bloodmeal. DAPI-stained nuclei appear blue. (Scale bar, ≈100 μm.) (d) Comparative immunoblot analysis of whole midgut lysates from A. stephensi (AS), A. arabiensis (AA), A. freeborni (AF), and A. gambiae (AG) with α-AgAPN1 PAb suggest that α-AgAPN1 PAbs recognizes a conserved midgut molecule across different malaria insect vectors. (e) Immunoblot analyses of glycoproteins in fractions eluted from the jacalin column with the Gal concentration (mM) indicated at the top of each lane. Blots were probed with α-AgAPN1 PAb (Upper) or the anti-glycan mAb MG96 (9) (Lower). MV, microvilli-enriched fraction (column input). Upper arrowhead indicates the predicted full-length AgAPN1 detected by both antibodies, whereas the lower arrowhead indicates protein doublet recognition specific for α-AgAPN1 PAb. MG96 recognizes a protein doublet (double arrowhead) with a different mobility relative to size markers than the doublet in Upper. Position of migration of molecular mass markers (kDa) is shown on the left of each immunoblot.