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. 2018 Sep 20;131(18):jcs220251. doi: 10.1242/jcs.220251

Fig. 3.

Fig. 3.

EGFR–LexAVP16 does not activate transcription when expressed at physiological levels. (A) The sgRNAs used to edit the endogenous Egfr sequence (lower) and the construct used for homology-directed repair (upper). (B) Western blot of lysates from wild-type and homozygous Egfr-LexAVP16 CRISPR adult flies and from ap-GAL4; UAS-EGFRLexAVP16 larval wing discs. In the CRISPR flies, EGFR is expressed at wild-type levels and migrates at the size for EGFR–LexAVP16. The wing disc extracts had much stronger EGFR–LexAVP16 expression and less protein was loaded. (C) Egfr-LexAVP16 eye disc stained with anti-LexA (C) and anti-EGFR (C′) antibody. Scale bar: 50 μm. (D,E) Eye disc (D) and wing disc (E) with clones lacking the Egfr-LexAVP16-modified locus marked by the absence of RFP (red) and LexA (D′,E′, blue). Anti-GFP antibody staining shows LexAop:6XGFP expression (D″,E″, green), which is not detectable in cells with Egfr-LexAVP16 above the background staining present in wild-type cells. Scale bars: 50 μm. (F–H) Egfr-LexAVP16; LexAop:FLP; Act>CD2>GAL4 adult head (F), ovary (G) and testis (H) showing no expression of UAS-6×GFP stained with anti-GFP antibody (green, F) or of UAS-mCherry-NLS (magenta, G,H). (I) A negative control adult head without Egfr-LexAVP16. (J,K) nSyb-LexAp65; LexAop:FLP; Act>CD2>GAL4 induces strong expression of UAS-6×GFP in adult brain (J) and of UAS-mCherry-NLS in larval brain (K). F–J are stained for Discs large (Dlg, magenta in F,I,J; green in G,H) and K is stained for E-cadherin (Ecad, green). Scale bars: 100 μm.