Table 1.
Voltage regime for isoelectric focusing.
Figure 1.
Staining patterns of mAbs aa2 and ab52.
(A) Cryosections of an adult fly head were probed with mAb aa2 (left) and mAb ab52 (right). Both antibodies stain all synaptic neuropil (green) but not the surrounding the cell body layer whose nuclei are stained with DAPI (blue). (B, C) Synaptic boutons of larval motor neuron terminals stained with anti-HRP (left, red) and mAbs aa2 (B, middle, green), or ab52 (C, middle, green). The overlays in the right column demonstrate that the epitopes recognized by both mAbs are present in all boutons (here shown for muscles M12/13 (B) and muscles M6/7 (C)) but not in the axons. Scale bars in A: 100 µm; in C for B and C: 50 µm)
Figure 2.
mAbs aa2 and ab52 recognize soluble proteins at identical Mr on 1D Western blot.
(A) Blot of a single SDS gel electrophoresis lane loaded with homogenate from 2 wild-type (CS) heads. The blotted membrane was vertically cut in two halves; one was developed with mAb aa2 (left), the other with mAb ab52 (right). Signals at identical Mr suggests that both mAbs probably recognize the same antigen. (B) Western blot of subcellular fractions of wild-type (CS) fly heads showing that the protein detected by mAb ab52 is exclusively present in the cytosolic supernatant. S1 = postnuclear supernatant; S2 = cytosolic fraction; P2 = total membrane fraction (10 head equivalents loaded per lane). The synaptic vesicle protein CSP recognized by the mAb ab49 was used as a marker for the total membrane fraction.
Figure 3.
aa2 and ab52 produce signals at identical Mr and pI on 2D Western blot profile.
Two sequential Western blots from a single 2DE gel loaded with sample equivalent to 25 fly heads. The two membranes were cut along the horizontal white line, the upper parts were developed with mAbs aa2 (left, dilution 1∶2) or ab52 (right, dilution 1∶10), the lower parts were stained with mAbs 3C11 (anti-SYN, 1∶100) and nc46 (anti-SAP47, 1∶200) as controls for both blots.
Figure 4.
Identification of mAb aa2 antigen by comparison of a Coomassie stained gel and a Western blot.
Coomassie-blue stained gel (left) and Western blot of a gel run in parallel (right). Loaded was: supernatant S1 (3 head equivalents, lanes 1, 1′), immunoprecipitation of S1 with mAb aa2 and protein-G beads (∼80 head equivalents, lanes 2, 2′ or ∼800 head equivalents, lanes 3, 3′), and 3 heads homogenized in Lämmli buffer (lanes 4, 4′). The blot was developed with mAb aa2. LC, light chain, HC, heavy chain of mAb aa2. The boxed bands of the gel were cut out and subjected to MS analysis.
Figure 5.
Enrichment of the ab52 antigen for MS by NEPHGE/SDS-PAGE and its final identification as Eps15.
(A) Western blot developed with mAb ab52 after partial transfer of proteins from the gel shown in (B). The signal from the mAb ab52 can be clearly seen as a distinct spot ∼100 kDa (asterisk), with a corresponding strong signal in the 1D lane loaded with 10 freshly homogenized fly heads. Non-specific signals were numbered as landmarks 1′–6′. (B) Silver stained NEPHGE gel with protein spots corresponding to the non-specific Western signals of (A) numbered 1–6 and the spot corresponding to the Western signal from mAb ab52 (encircled). (C) Western Blot developed with mAbs ab52 and ab49 showing that in the Eps15 null mutant eps15Δ29 the ∼100 kDa signal characteristic for ab52 in the wild type (CS) is absent. CSP recognized by mAb ab49 was used as a loading control.
Table 2.
Proteins with cumulative Mascot scores>300 identified in the gel pieces boxed in lane 2 and 3 of Fig. 4.
Figure 6.
Verification of the prime candidate from MS, EPS15, as the antigen recognized by mAbs aa2 and ab52.
Immunohistochemical staining of larval synaptic neuropil with mAbs aa2 (A) and ab52 (B) is present in wild type (WT) but absent in eps15Δ29 null mutants (Δ29) and perfectly matches the distribution of Eps15 in synaptic boutons, here shown on muscle M13 (C). Scale bar in A for A and B: 100 µm; in C: 5 µm.
Table 3.
Proteins identified in the gel piece encircled in Fig. 5B.