Figure 4. Capture and identification of newly-synthesized proteins from neurons and glial.

(A) In-gel AF488 fluorescence across a broad range of proteins in fly brain extracts expressing penicillin G acylase (PGA) pan-neuronally and following incubation with phenylacetyl-OPP (PhAc-OPP). Controls groups are no PGA expression (with PhAc-OPP) or no PhAc-OPP (with PGA expression). (B) Detection of total desthiobiotin-tagged protein in brain lysates (input) or pulldown fractions (eluate) following pan-neuronal PGA expression, PhAc-OPP incubation, conjugation of OPP-labeled protein to desthiobiotin azide and neutravidin bead pulldown. A strong background band of unknown endogenously biotinylated protein is observed between 100–150 kDa. (C) Enrichment of neuronal proteins Brp, Synapsin, and Syntaxin following neutravidin pulldown of desthiobiotin-tagged protein from pan-neuronal elav-GAL4/UAS-PGA brain extracts. (D) Pan-glial PGA expression (Repo-GAL4/UAS-PGA) promotes broad O-propargyl-puromycin (OPP) protein labeling in brain extracts following PhAc-OPP incubation but not vehicle control. Enrichment of total desthiobiotin-tagged protein seen following neutravidin bead pulldown of the glial proteome, detected with anti-biotin (D) and enrichment of the glial-specific protein Draper (with apparent mol. weight shift between input and eluate) from Repo-GAL4/UAS-PGA extracts, but not the neuron-specific protein Syntaxin. Data are representative of three independent experiments.