Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen -- Dinglasan et al. 104 (33): 13461 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Dinglasan et al. 10.1073/pnas.0702239104.

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SI Figure 4
SI Figure 5
SI Figure 6
SI Table 2

SI Figure 4

Fig. 4. Immunoblot analysis using anti-AgAPN1 polyclonal antibodies. (a) Preimmune serum recognition of recombinant AgAPN1 protein (rAgAPN1), brush border microvilli (BBMV), and whole midgut (MG) antigens from Anopheles gambiae (Ag). Arrow indicates background recognition of the recombinant protein. Anti-AgAPN1 antibodies did not recognize AgAPN1 in female non-gut tissues. (b) Immunoblot analysis of supernatant (SP) and pellet (PL) fractions of glycoproteins after treatment of the A. gambiae microvilli-enriched suspension (MV) with Bacillus cereus phosphotidylinositol-specific phospholipase C. C, mosquito carcass (whole body minus midgut). The migration of molecular mass markers (kDa) are shown on the left.

SI Figure 5

Fig. 5. Anti-AgAPN1 polyclonal antibodies block Plasmodium falciparum development in A. gambiae mosquitoes. A. gambiae mosquitoes were allowed to feed on P. falciparum-infected blood along with increasing concentrations of AgAPN1 PAb [IgG, mg/ml]. Control mosquitoes were fed on infected blood along with 1.0 mg/ml of preimmune rabbit IgG. Horizontal bar and no. indicate the median oocyst number per mosquito midgut for each IgG treatment group. Percentage inhibition (I) = [(control median oocyst no. [minus] experimental median oocyst no.)/control median oocyst no.)] [times] 100. Significance values were determined by Mann-Whitney U test (a = 0.05).

SI Figure 6

Fig. 6. RT-PCR and immunoblot expression analysis of AgAPN1 after dsRNA injection into A. gambiae female mosquitoes. (a) RTPCR showing the absence of midgut gene silencing between 48 and 96 h after inoculations with 800 ng of dsAgAPN1. The number of cycles is shown in parentheses on the right. Amplification of the Anopheles S7 ribosomal gene was used as loading control. (b) Immunoblot analysis of midguts from the same cohort of mosquitoes that received 800 ng of dsAgAPN1 (a Upper). (c) Immunoblot analysis of midguts from 72-h and 96-h time points postinjection with 5,400 ng of dsAgAPN1. Molecular mass markers (in kDa) are shown on the far left. Midguts from mosquitoes that were injected with equivalent amounts of dsGFP were used as a control (CTRL). Each lane of the gel was loaded with 25 mg of protein from a pool of 10 mosquito midguts per time point.

Table 2. Primer sequences for the production of A. gambiae AgAPN1 dsRNA molecules

Primer	Sequence	Region of the gene
1-Forward	TAATACGACTCACTATAGGG ATTCGCTTGGCTGCTCGAACAAT	3' exons
2-Forward	TAATACGACTCACTATAGGG GAACGCTACCGTCTGCCAACAA	5' exons
3-Forward	TAATACGACTCACTATAGGG TGGTGGTGTTGCGAGTGATGAGAT	5' UTR
4-Forward	TAATACGACTCACTATAGGG CTAATGATCTTGATCCCGGGTACT	3' UTR
1-Reverse	TAATACGACTCACTATAGGG TTATCCCATGAGCAGGTGAACCGT	3' exons
2-Reverse	TAATACGACTCACTATAGGG TGCCAGATATCTTCGCTCGCCATT	5' exons
3-Reverse	TAATACGACTCACTATAGGG CAACGCAATCTTTGACCGCCAA	3' UTR

T7 promoter is shown in bold. A mixture of dsRNAs corresponding to different fragments of the transcript were used to knock down AgAPN1 expression in the midgut.