4.3 Results
4.3.2 Interactions in brain lysate
To test for binding between Arc and candidate interactors, we performed GST pull- down experiments. GST-Arc was immobilized on glutathione-agarose beads which were then incubated with DOC-solubilized brain-membrane fraction. The presence of candidate interactors was measured by immunoblot. To assess whether binding
61 67 71
% full length
Purified GST-Arc Depleted once Depleted twice full length GST-Arc
GST-Arc fragment
Figure 4.2: Brief incubation with glutathione (GSH) agarose beads depletes the GST-Arc fragment. GST-Arc expressed in E. coli is purified by GSH-agarose in- cubation and elution and analyzed by SDS-PAGE stained for total protein. The two most prominent species in the solution are full-length GST-Arc and a GST-Arc fragment of approximately 30 kiloDaltons (lane one). The fragment is depleted by an additional five-minute incubation with GSH-agarose beads; the supernatant is retained (lane 2). An additional incubation results in a further depletion of the fragment (lane 3). The depletion is the result of the affinity of GSH-agarose beads for the GST-Arc fragment, which is higher than the affinity of the beads for the full-length GST-Arc protein.
was specific to Arc, we compared the amount of protein pulled down by GST-Arc to that pulled down by GST alone.
To compare equimolar amounts of our bait proteins, GST and GST-Arc, we gener- ated multiple samples by serially diluting our immobilized bait, measured the amount of bait protein in each sample, and selected equimolar samples for comparison. Specif- ically, we diluted bait-incubated GSH-agarose beads with non-incubated GSH-agarose beads (described in Materials and Methods section 4.2.3, shown in Figure 4.3). We analyzed the amount of bait protein in each sample using SDS-PAGE and total pro- tein stain, measuring the relative mass of each bait species via densitometry. This
number was converted to a molar intensity by dividing the mass by the molecular weight of the species in kilodaltons (kDa).
Table 4.1 contains the densitometry measurements for the bead samples shown in Figure 4.3. In this example, the relative number of moles of GST in an aliquot composed only of beads incubated with GST is 26 (shown in the first column of the table, the GST sample), and a comparable molar amount of GST-fusion species is present in an aliquot composed only of beads incubated with GST-Arc (28; the sum of the molar intensity value listed for columns 4 and 6, full-length GST-Arc and GST-Arc fragment).
Table 4.1: Quantification of the mass and molar amounts of bait protein species in a pull- down experiment.
full-length GST-Arc
GST GST-Arc fragment
Percentage of beads
incubated with bait 100 50 25 100 50 100 50 Integrated intensity 684 374 162 314 195 713 447
Molar intensity 26 14 6 4 2 24 15
Figure 4.4 shows results from a pull-down experiment. SDS-PAGE lanes were loaded with the same samples described in Figure 4.3. The first lane contains the input, DOC-solubilized brain-membrane fraction, which contains the candidate inter- actors. Lanes 2–4 contain decreasing concentrations of GST immobilized on agarose and incubated with the input. Lanes 5–6 contain decreasing concentrations of GST- Arc immobilized on agarose and incubated with the input. Lanes 2 and 5 can be nearly directly compared; as indicated in Table 4.1, the relative number of moles of
Bait Protein Bait Protein Bait Protein Bait Protein Bait Protein
input GSTGSTGST GST-ArcGST-Arc
% loaded beads
% loaded beads 100 50 25 100 50
GST
Full length GST-Arc
GST-Arc fragment
Figure 4.3: Total protein as visualized by SDS-PAGE allows selection of equimolar conditions. Samples from a GST-Arc pull-down experiment were analyzed: 1) DOC- solubilized brain-membrane fraction (input), which contains the potential interac- tors; 2-4) GST immobilized on glutathione-agarose beads, diluted to the percent indicated with fresh glutathione-agarose beads, incubated with input; 5-6) GST-Arc immobilized on glutathione-agarose beads, diluted to the percent indicated with fresh glutathione-agarose beads, incubated with input. Full-length GST-Arc (solid black arrowhead) is approximately 80 kDa; a prominent GST-Arc fragment (empty arrowhead) is approximately 30 kDa.
GST species is 26 in the GST-only conditions and 28 in the GST-Arc “100% loaded beads” conditions, with GST-Arc fragment comprising 26 of the 28 relative moles.
Thus the bands of each interactor can be compared between the “100% loaded beads”
lanes in this experiment, at least within an error range of 10% (a generous estimate of the difference in binding, possibly resulting from a 26:28 molar ratio).
The greater intensity of the CaMKIIα band in the GST-Arc 100 lane compared to the GST-100 lane indicates that more CaMKII bound GST-Arc than GST. Because the GST-Arc beads are only 1/7th as long as full-length GST-Arc (4/28; Table 4.1), the difference we see may be only 1/7th of the difference of CaMKII binding to GST- Arc and GST alone. This assumes CaMKII does not bind a region of Arc contained in the GST-Arc fragment, which contains approximately the first 40 amino acids of Arc.
Although we probed for CaMKIIα, the signal does not exclusively represent an in- teraction between CaMKIIαand Arc. First, because this pull-down was performed in brain-membrane fraction, an interaction between CaMKII and Arc could be mediated by an adapter protein. Secondly, the typical composition of a CaMKII holoenzyme in the forebrain is approximately three α subunits to one β subunit. An interaction with with either could be detected with the anti-CaMKIIα antibody.
Comparison of the GluR1 and SynGAP bands in this experiment shows that both of them bound more to GST-Arc than GST in this experiment. Since PSD-95 bands were not observed, the GST-Arc beads did not pull down large portions of the postsynaptic density (PSD).
Repeating this optimized experiment several times indicated an interaction be- tween Arc and CaMKII at p<.05, and a trend indicating that Arc bound SynGAP and GluR1. However, the immunoblot bands were faint, and background was high, which added uncertainty to the quantification. Changes to the number and duration of bead wash steps decreased background, but also decreased signal. When exper-
iments implementing these changes were included, no interactions were statistically significant.
CaMKII!
GluR1 SynGAP
PSD-95
Bait Protein Bait Protein Bait Protein Bait Protein Bait Protein
input GSTGSTGST GST-ArcGST-Arc
% loaded beads
% loaded beads 100 50 25 100 50
Figure 4.4: Immunoblot GST-Arc pull-down from brain-membrane fraction; not representative. Glutathione-agarose beads were incubated with GST or GST-Arc as described and subsequently incubated with deoxycholate (DOC)-solubilized brain- membrane fraction. After incubation, the beads were washed and loaded on to an SDS-PAGE gel, where the proteins were eluted with sample loading buffer. Input was also loaded on the gel. Immunoblots were probed for CaMKII, GluR1, SynGAP and PSD-95. GST-coated beads incubated with input were loaded in lanes 2 through 4; GST-Arc coated beads incubated with input were loaded in lanes 5 and 6. The percent loaded beads indicates whether the loaded beads were diluted with newly swollen glutathione beads. Relative enrichment was calculated as described in the text. This blot shows binding between Arc and CaMKII, and a small increase over background binding with GluR1 and SynGAP. However the results were inconsistent and were not present when we made changes to the post-incubation bead wash to reduce background.