Figure 2: Tagging of ANL-labelled proteins in Drosophila larvae and flies.
From: Cell-selective labelling of proteomes in Drosophila melanogaster
L3 stage larval body walls (a) or brains of L3 stage larvae (d) were dissected after chronic ANL feeding using 4 mM ANL concentration. Heads from adult Drosophila flies (b,c) were collected 0–3 days post eclosion after chronic ANL feeding using an ANL concentration of 4 mM. ANL is efficiently incorporated into muscle (a), neuronal (b) or glial proteins (c) when Drosophila larvae (a,d) or adult flies (b,c) express mMetRSL274G-EGFP or dMetRSL262G-EGFP. ANL-labelled proteins were tagged by a biotin-alkyne affinity-tag, and purified via NeutrAvidin agarose. Tagged input (I, before NeutrAvidin purification), unbound (U, no ANL-containing proteins) and eluted (E, enriched ANL-labelled proteins after NeutrAvidin purification) fractions from ANL labelled and control samples were run in mirror-imaged order on SDS-PAA gels, blotted and probed with anti-biotin antibody. Effective ANL labelling and subsequent biotin tagging were verified for selected marker proteins (‘anti-candidate protein’), that is, Dlg (a) in muscles, Synapsin in neurons (b) as well as Draper I (c) and to a small amount Dlg (c) in glia cells. Furthermore, the intracellular domain (d) of the transmembrane protein Notch was found to be ANL labelled when dMetRSL262G-EGFP is expressed ubiquitously.
