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. 2015 Apr 27;11(4):e1004848. doi: 10.1371/journal.ppat.1004848

Fig 2. The high expression of AaHig in the brain of A. aegypti.

Fig 2

(A-B) The expression of AaHig in various tissues of A. aegypti. The abundance of AaHig was assessed via SYBR Green qPCR (A) and immuno-blotting with an AaHig antibody (B). (A) The total RNA was isolated from mosquito tissues to determine AaHig expression by SYBR Green qPCR and normalized by A. aegypti actin (AAEL011197). The qPCR primers were described in the S1 Table. Data were represented as the mean ± standard error. (B) A variety of tissues were dissected from female A. aegypti. 50 μg of total protein from tissue lysates was loaded into each lane. The detection of A. aegypti actin was used as the internal control. (C) Secretory property of AaHig. The full length of AaHig gene (1bp-2436bp) was inserted into the expression vector pAc5.1/V5-His A with a V5 tag. The recombinant DNA plasmid (pAc-AaHig) was transfected into S2 cells. AaHig expression was detected with anti-V5 antibody in the cell lysate and supernatant. (D) AaHig staining in A. aegypti Aag2 cells. Aag2 is an A. aegypti cell lineage of embryonic origin. The pAc-AaHig recombinant plasmid was transfected into Aag2 cells. AaHig was stained by anti-V5 antibody and anti-mouse IgG Alexa-546 (Red). The plasma membrane was stained by the Wheat Germ Agglutinin (WGA) conjugated with Alexa-488 (Green). Nuclei were stained with To-Pro-3 iodide (Blue). Images were examined using the 63×objective lens of a Zeiss LSM 780 meta confocal. (E) AaHig is localized on the cell surface of neural cells of the A. aegypti brain. The brains were dissected from female A. aegypti for the in situ staining. An anti-HRP rabbit polyclonal antibody with anti-rabbit IgG Alexa-488 was used for surface staining of mosquito neural cells (Green). The AaHig protein was detected by an anti-AaHig mouse polyclonal antibody and anti-mouse IgG Alexa-546 (Red). Nuclei were stained blue with To-Pro-3 iodide. Images were examined using the 10× and 63×objective lens of a Zeiss LSM 780 meta confocal. AL, antennal lobes; OL, optic lobes.