DEV018101 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Affinity purified polyclonal rabbit α-Numb recognizes endogenous LvNumb. Injection of LvNumb morpholino quenches the expression of the LvNumb 5′UTR-GFP reporter construct, and manipulation of Notch signaling does not affect expression of LvNumb. (A) Western analysis of mesenchyme blastula protein extracts using rabbit pre-immune sera and affinity purified rabbit α-LvNumb pAb. Arrowhead indicates a band recognized by the antibody that is the predicted size of full-length LvNumb (67 kDa). (B) QPCR analysis of LvNumb expression from samples of embryos injected with LvN^act or LvN^neg mRNA. Neither affects significant changes in transcription of lvnumb. ΔCT values must show a 1.7 cycle difference from controls to be considered significant. (C) Bright-field images overlaid with fluorescent GFP images of midgastrula embryos injected with lvnumb 5′UTR-GFP mRNA and control morpholino. (D) Brightfield images overlayed with fluorescent GFP images of midgastrula embryos injected with lvnumn 5′UTR-GFP mRNA and LvNumb1 morpholino. (E) Control mesenchyme blastula embryo expresses lvnumb transcripts. (F) Mesenchyme blastula embryo injected with LvN^NEG expresses lvnumb transcripts. (G) Control mesenchyme blastula stage embryo stained anti-Notch antibody (red) showing clearance of the Notch receptor for the NSM (between white bars). (H) The Notch receptor is still cleared in embryos injected with LvNumb morpholino (between white bars). (I) Control pluteus with pigment cells. (J) LvNumb embryo from same experiment as in H fails to produce pigment cells.