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. 2013 Jan 1;140(1):176–184. doi: 10.1242/dev.088799

Fig. 2.

Fig. 2.

Notch expression and endocytic trafficking are altered in Abl clones. (A-D′) Eye and (E) wing disks with Abl clones stained with anti-Notch intracellular domain (NID, red) and anti-GFP (green). All images are maximal projections. (A,A′) A slight increase in punctate Notch staining is detected in Abl clones in a third instar disk. (B,B′) In 24 hours APF disks, Notch staining is slightly more pronounced with some larger puncta (yellow arrows) in the Abl clones than in wild-type tissue. (C,C′) By 48 hours APF, the increased Notch staining in Abl clones relative to wild type is striking. (D,D′) At 72 hours APF, Notch staining appears absent in the wild-type tissue but is present ectopically in Abl mutant clones. (E-E″′) In third instar wing disks, more and bigger Notch puncta are evident in Abl clones than in wild type. E″ and E″′ are magnified views of the regions boxed in E and E′. (F-K′) Live 24 hour APF eye disks carrying Abl clones were pulse labeled with antibody recognizing the extracellular domain of Notch (anti-ECN, Red), washed, and either fixed immediately (F,I) or chased for 30 (G,J) or 60 (H,K) minutes to follow Notch trafficking. Detergent was added to permeabilize the tissue for visualization of internalized Notch protein. (F-H) Single optical sections across the middle plane of the epithelium. (I-K) Orthogonal sections of the same disk shown in F-H, respectively, with apical and basal surfaces marked by arrows on the right. (F) Internal Notch staining is absent with no chase, indicating all labeled receptor is at the cell surface. (G) After 30 minutes chase, some Notch puncta are seen in Abl clones. (H) After 60 minutes chase, more and brighter Notch puncta are evident in Abl clones than in wild-type. (I) With no chase, most Notch localizes to the apical-junctional surface, with little or no detectable cytoplasmic staining. (J) After 30 minutes chase, Notch is barely detectable at the apical-junctional surface, while cytoplasmic puncta become evident. (K) After 60 minutes chase, brightly stained Notch puncta are apparent in Abl clones. Scale bars: 10 μm. A, apical; B, basal.