mouse anti-eys/spam (21a6)  (Developmental Studies Hybridoma Bank)

Journal: Journal of cell science

Article Title: Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors.

doi: 10.1242/jcs.091223

Figure Lengend Snippet: Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) (A,B), Rh1 (C,D), Spam (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).

Article Snippet: The following primary antibodies were used: rat anti-Crb2.8 antibody (1:1000) (Richard et al., 2006), rabbit anti-Sdt-PDZ antibody (1:500) (Berger et al., 2007), mouse anti-Sdt-PDZ antibody (B1-8, 1:200) (Bulgakova et al., 2010), rabbit antiDrosophila-PATJ antibody (1:500) (Richard et al., 2006), rat anti-DE-Cad antibody (DCAD2, 1:50, DSHB), mouse anti-Na+/K+-ATPase antibody (a5, alpha subunit, 1:500 concentrate, DSHB), rabbit anti-Rh1 antibody (1:1000) (Satoh et al., 2005), mouse anti-Rh1 (4C5, 1:100, DSHB), mouse anti-Spam antibody (21A6, 1:100, DSHB) (Zelhof et al., 2006), mouse anti-FLAG M2 antibody (1:1000, Sigma), rabbit anti-FLAG antibody (1:200, Sigma, F7425), rabbit anti-Baz antibody (1:400, kindly provided by Andreas Wodarz) (Wodarz et al., 1999), guinea pig anti-Par-6 antibody (1:1000, kindly provided by Andreas Wodarz) (Kim et al., 2009), mouse anti-Arm antibody (N2 7A1, 1:50, DSHB), rabbit anti-PKC f C20 (1:1000, Santa Cruz Biotechnology), rabbit anti-phosphorylated-Ezrin/Radixin/Moesin (1:100, Cell Signalling Technology), and rabbit anti-bH-spec (1:500, kindly provided by Graham Thomas) (Thomas and Kiehart, 1994).

Techniques: Over Expression, Staining, Expressing