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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: Dev Cell. 2008 Oct;15(4):617–626. doi: 10.1016/j.devcel.2008.08.017

Figure 2. Spn77Ba is Expressed by Tracheal Cells and is Localized at Apical Surface of Tracheal Epithelium Where Melanization is Initiated.

Figure 2

(A) A Gal4 driver containing 2.2 kbp of DNA from 5’end of Spn77Ba gene was used to activate GFP expression from a UAS-GFP construct. GFP signal is seen in trachea and at lower levels in gut and epidermis.

(B) When expressed by transfected S2 cells, HA-tagged Spn77Ba protein was detected mainly in the culture medium by Western blot using anti-HA antibody.

(C-D) When Spn77Ba-GFP fusion protein was expressed under control of the da-Gal4 driver, GFP signal (C and D, green) was seen on apical surface of tracheal epithelium, toward luminal side of apical F-actin and co-localized with chitin. F-actin of tracheal cells was stained with Alexa Fluor 633–phalloidin (C’, red) and chitin was visualized with a rhodamine-conjugated chitin-binding probe (D’, red). N: nucleus.

(E) Melanization (arrow) resulting from Spn77Ba RNAi can be initially detected between tracheal cuticle and epithelial cells. Cells of tracheal epithelium were visualized by expression of cytoplasmic GFP under control of the btl-Gal4 driver (E’). N: nucleus.

(F) Non-melanized and melanized sections of trachea in btl>Spn77Ba-IR larva were compared for integrity of the basement membrane, which was visualized with a vkg-GFP reporter (Morin et al., 2001). F-actin staining with Alexa Fluor 633–phalloidin was used to visualize the epithelial cells.