Journal: eNeuro

Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

doi: 10.1523/ENEURO.0175-17.2017

Figure Lengend Snippet: CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with >). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with >). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.

Article Snippet: The following antibodies were used for immunocytochemistry: extracellular epitopes of GluA1 (Alomone Labs, catalog #AGC004), HA-tag [BioLegend, 16B11, catalog #901512 (mouse)], HA-tag [Santa Cruz Biotechnology, Y-11 (rabbit), catalog #SC-805], and synaptophysin (Sigma-Aldrich, catalog #SAB4502906).

Techniques: Staining

Journal: eNeuro

Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

doi: 10.1523/ENEURO.0175-17.2017

Figure Lengend Snippet: CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p < 0.05.

Article Snippet: The following antibodies were used for immunocytochemistry: extracellular epitopes of GluA1 (Alomone Labs, catalog #AGC004), HA-tag [BioLegend, 16B11, catalog #901512 (mouse)], HA-tag [Santa Cruz Biotechnology, Y-11 (rabbit), catalog #SC-805], and synaptophysin (Sigma-Aldrich, catalog #SAB4502906).

Techniques: Infection, Western Blot