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. Author manuscript; available in PMC: 2008 Dec 1.
Published in final edited form as: Insect Biochem Mol Biol. 2007 Sep 4;37(12):1327–1337. doi: 10.1016/j.ibmb.2007.08.006

Figure 4. Expression of TH.

Figure 4

A) Northern blot analysis of TH demonstrates expression in eggs (day 3–4) and in larval ventral nerve cord and integument. Expression was not detected in fat body, hemocytes or midgut. Top panel: several hour exposure. Middle panel: 3 day exposure. Bottom panel: methylene blue staining of ribosomal RNA on blot demonstrating that similar amounts of RNA were loaded per lane. B) Immunoblot analysis of TH confirms expression in eggs, integument and ventral nerve cord. The lower molecular weight polypeptide in the VNC sample may be encoded by an alternatively spliced TH mRNA or may be a degradation product. The lower molecular weight polypeptide in the fat body sample is probably caused by the interaction of the polyclonal antiserum with an unknown protein because a second polyclonal antiserum generated against TH does not recognize this unknown protein (data not shown). Note that proPO (bottom panel) was abundant in hemocytes and present in plasma. The source of proPO detected in other tissues is likely to be adherent hemocytes. C) Immunoblot analysis illustrates higher abundance of TH in prepupal segments that were just starting to tan (third thoracic and first abdominal) compared with one that was not (second thoracic). Note that proPO was present in approximately equal amounts in each of these samples. The bottom panel shows the anterior part of a newly emerged pupa. The highly tanned metathoracic bar and the partially tanned first abdominal segment correlate with the presence of TH. D) Semiquantitative RT-PCR demonstrates expression of TH in the hemocytes and fat body of three larvae 24 hours after they were inoculated with bacteria (I1–3). TH PCR products from control larvae (C1-3) were barely detectable in this experiment in which 25 PCR cycles were performed. TH expression in the hemocytes and fat body of naive larvae was detectable when the number of PCR cycles was greater (not shown). PPO1 and PPO2 were not upregulated by the immune challenge. Rps3 was used as a template concentration control gene. E) Immunoblot analysis confirmed expression of TH in the hemocytes and fat body of infected larvae (I1-2) but not control larvae (C1-2). No TH was detected in plasma. In contrast, the concentration of proPO did not increase in response to inoculation.