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. 2019 Sep 30;8:e48750. doi: 10.7554/eLife.48750

Figure 1. Schematic of nAb generation and validation pipeline.

(A) Schematic of nAb generation pipeline. See text for details. (B) Schematic of pipeline for validating ELISA-positive nAbs for utility as intrabodies and immunolabels.

Figure 1.

Figure 1—figure supplement 1. Anti-Homer1 nAbs that function as intrabodies colocalize with exogenously expressed Homer1 when coexpressed in heterologous COS-1 cells.

Figure 1—figure supplement 1.

Representative images of the intrabody positive anti-Homer1 nAb HS22 validated in the heterologous COS-1 cell intrabody assay. (A) Images of HS22-EGFP (green) and Homer1 (red) expressed alone in COS-1 cells. The scale bar on the top left panel is 10 μm and holds for all panels in the figure. (B) Images of HS22-EGFP (green) and Homer1 (red) coexpressed in COS-1 cells and demonstrating the loss of HS22-EGFP nuclear localization and its cytoplasmic colocalization with coexpressed Homer1. The graph shows the normalized fluorescence intensity values across the line scan depicted by the white line in the merged image, with the corresponding R2 value. (C) Images of EGFP (green) and Homer1 (red) coexpressed in COS-1 cells and demonstrating the lack of ran effect of Homer1 coexpression on the nuclear localization of EGFP. The graph shows the normalized fluorescence intensity values across the line scan depicted by the white line in the merged image, with the corresponding R2 value. Note the concordance of the red and green intensity values for HS22-EGFP and Homer1 but not EGFP and Homer1.
Figure 1—figure supplement 2. nAbs that function as intrabodies against excitatory synaptic target proteins colocalize with the endogenously expressed target proteins in cultured hippocampal neurons.

Figure 1—figure supplement 2.

Representative images of intrabody-positive nAbs bound to endogenous excitatory synapse targets after expression in cultured hippocampal neurons. (A) The top left panel shops the pattern of EGFP expression in the dendrites of a transfected neuron. The subsequent rows show neurons expressing nAb-EGFP fusions and showing EGFP fluorescence (green), target-specific mAb labeling (magenta), and the merged image, with an adjacent panel showing a magnified image of the inset marked by the dashed box. Panels to the far right of each row are the normalized fluorescence intensity values across the individual line scans from the magnified inset, with the corresponding R2 values. Note the concordance of the magenta and green intensity values for the target and the positive nAb-EGFP fusions, but not for EGFP itself. The scale bar in the top left panel is 5 μm and holds for all panels in figure except the magnified insets, for which the scale bar is 2 μm. (B) Graph of Pearson’s Correlation Coefficient (PCC) values between nAb-EGFP and anti-target mAb labeling (Homer1: L113/130; IRSp53: L117/1; pan-SAPAP:N459/94). PCC values of Homer1 and IRSp53 mAb immunolabeling with EGFP fluorescence in cells expressing EGFP are also shown. (C) Size analysis of mAb-labeled puncta of target proteins between nAb-EGFP transfected and untransfected cells. Homer1: ns, p=0.3828; IRSp53: ns, p=0.5410; pan-SAPAP: ns, p=0.0706; two-tailed unpaired t-tests. Bars on all graphs are mean ± SD.
Figure 1—figure supplement 3. nAbs that function as intrabodies against inhibitory synaptic and ER-PM junction target proteins colocalize with endogenously expressed target proteins in cultured hippocampal neurons.

Figure 1—figure supplement 3.

Representative images of intrabody-positive nAbs bound to endogenous targets after expression in cultured hippocampal neurons. (A) The top row shows the diffuse localization of EGFP in the dendrite (left) and soma (right) of an EGFP-transfected neuron. The subsequent rows show neurons expressing nAb-EGFP fusions and showing EGFP fluorescence (green), target-specific mAb labeling (magenta), and the merged image, with an adjacent panel showing a magnified image of the inset marked by the dashed box. The scale bar in the top left panel is 5 μm and holds for all panels in figure except the magnified insets, for which the scale bar is 2 μm. Panels to the far right of each row are the normalized fluorescence intensity values across the individual line scans from the magnified inset, with the corresponding R2 values. Note the concordance of the nAb (green) and anti-target mAb (magenta) intensity values. (B) Graph of PCC values between nAb-EGFP fusions and anti-target mAb labeling (Gephyrin: L106/23; AMIGO-1: L86A/37). PCC values of Gephyrin and AMIGO-1 mAb immunolabeling with EGFP fluorescence in cells expressing EGFP are also shown. (C) Size analysis of mAb labeled puncta of target proteins between nAb-EGFP transfected and untransfected cells. Gephyrin: ns, p=0.8758; AMIGO-1: ns, p=0.1091; two-tailed unpaired t-tests. Bars on all graphs are mean ± SD.
Figure 1—figure supplement 4. nAbs that function as intrabodies against excitatory synaptic target proteins colocalize with the excitatory synaptic marker PSD-95 in cultured hippocampal neurons.

Figure 1—figure supplement 4.

(A) Each row shows representative images of a nAb against an excitatory synaptic protein (Homer1L, IRSp53, SAPAP2) expressed in neurons as nAb-EGFP fusions (green), and immunolabeling for a mAb (K28/43) against the synaptic marker PSD95 (magenta). The scale bar in the top left panel is 5 μm and holds for all panels in figure except the magnified insets, for which the scale bar is 2 μm. Panels to the far right of each row are the normalized fluorescence intensity values across the individual line scans from the magnified inset, with the corresponding R2 values. Note the concordance of the intensity values for the nAb-EGFP fusions (green) with PSD-95 labeling (magenta). (B) Graph of PCC values between the different nAb-GFP fusions and EGFP with immunolabeling for anti-PSD-95 mAb K28/43. (C) Size analysis of mAb labeled puncta of PSD-95 between nAb-EGFP transfected and untransfected cells. Homer1: ns, p=0.2093; IRSp53: ns, p=0.7063; pan-SAPAP: ns, p=0.2683; two-tailed unpaired t-tests. Bars on all graphs are mean ± SD.
Figure 1—figure supplement 5. nAbs that function as intrabodies against inhibitory synaptic and ER-PM junction target proteins localize to their respective subcellular domains in cultured hippocampal neurons.

Figure 1—figure supplement 5.

Representative images of the colabeling for proteins related to targets of nAbs and expressed nAbs-EGFP in neurons. (A) Each row shows representative images of a nAb-EGFP fusions (green) against the inhibitory synaptic protein Gephyrin (top row) or the ER-PM junction protein AMIGO-1 (second row) and in magenta immunolabeling with mAbs that label synapses (the pan-synapsin mAb L125/129 that sees all synapsin isoforms,) or Kv2 channel-containing ER-PM junctions (mAb K89/34 against Kv2.1 to label. The scale bar in the top left panel is 5 μm and holds for all panels in figure except the magnified insets, for which the scale bar is 2 μm. Panels to the far right of each row are the normalized fluorescence intensity values across the individual line scans from the magnified inset, with the corresponding R2 values. Note the concordance of the intensity values for the nAb-EGFP fusions (green) with labeling for the compartment-specific markers (magenta). (B) Graph of PCC values between the different nAb-GFP fusions and EGFP with immunolabeling for synapses (L125/129) or ER-PM junctions (K89/34). (C) Size analysis of mAb labeled puncta. Left: synapses (labeled with anti-pan-synapsin mAb L125/126) in anti-Gephyrin nAb-EGFP transfected and untransfected cells: ns, p=0.7063. Right: Kv2.1-containing ER-PM junctions (labeled with anti-Kv2.1 mAb K89/34) in anti-AMIGO-1 nAb-EGFP transfected and untransfected cells: ns, p=0.6513. Two-tailed unpaired t-tests. Bars on all graphs are mean ± SD.
Figure 1—figure supplement 6. Anti-Homer1 nAbs exhibit labeling of exogenously expressed Homer1 in heterologous COS-1 cells.

Figure 1—figure supplement 6.

Representative images of anti-Homer1 nAb labeling (green) of transiently transfected COS-1 cells expressing Homer1L double-labeled with the anti-Homer1 mAb L113/27 (red). The left column shows a mosaic of transfected and untransfected cells, nuclei are stained with Hoechst dye in blue. Note overlap of nAb and mAb labeling at both the cellular and subcellular level. The scale bar in the top left panel is 50 μm and holds for all panels in column. The three panels to the right show higher magnification views of the cells labeled with the asterisk in the left panels. The scale bar in the top left panel is 5 μm and holds for all panels in column. The graph to the right shows the normalized fluorescence intensity values across the line scans depicted by the white line in the merged images, with the corresponding R2 values.