Fig. 1: Validating AMPA receptor labeling reagents derived from monoclonal antibodies.
From: Genetically encoded intrabody probes for labeling and manipulating AMPA-type glutamate receptors
a Approach for generating R-mAbs in HEK cells. b A1 scFv-Halo (cyan) binds to HEK cells expressing GFP-tagged GluA1 and GluA2 AMPAR subunits (top panels, 29/32 cells from 1 culture) but not control cells (GFP alone, bottom panels, 0/33 cells from 1 culture). Scale bar is 5 µm. c A2 Fab-Halo (cyan) binds HEK cells expressing GFP-tagged GluA1 and GluA2 AMPAR subunits (top panels, 27/35 cells from 2 cultures) but not control cells (GFP alone, bottom panels, 0/36 cells from 2 cultures). Scale bar is 5 µm. d Primary hippocampal neuron expressing saCas9 with a gRNA targeting GluA2 or saCas9 alone (no gRNA control) along with mCherry (red). White arrowheads indicate the transfected neurons, yellow arrowhead indicates a neighboring untransfected neuron. Scale bars are 10 µm. e Quantification of A2-Fab-Halo (left panel) or A1 scFv-Halo (right panel) labeling in cells expressing saCas9 along with GluA2 or GluA1-targeting gRNA (data are normalized to signal from control cells not expressing gRNA) (GluA2 control n = 11 cells, GluA2 gRNA n = 18 cells from 2 cultures, GluA1 control n = 11 cells, GluA1 gRNA n = 17 cells, from 1 culture, two-tailed Student’s t-test, ****p < 0.0001 for both GluA1 and GluA2). f Left: Hippocampal neurons labeled with designated R-mAb (cyan), designated AMPAR subunit antibody (magenta) and Homer1 (green). White arrowheads indicate colocalization of R-mAb signal with anti-Homer and respective anti-GluA1 or anti-GluA2 antibody. Scale bars are 10 µm; insets are 5 µm. g Quantification of A1 scFv-Halo (cyan), A2 Fab-Halo (red) and A2 scFv-Halo (magenta) JF635i signal stability, normalized to signal immediately following washout (t = 0) (A1 scFv-Halo n = 50 dendritic ROIs from 6 cells, A2 Fab-Halo n = 50 dendrite ROIs from 6 cells, A2 scFv-Halo n = 50 dendrite ROIs from 6 cells). h Mobility of AMPARs labeled with designated R-mAb, measured by fluorescence recovery after photobleaching. (A1 scFv-Halo n = 21 spines, A2 Fab-Halo n = 22 spines, A2 scFv-Halo n = 18 spines). Data are presented as mean values +/- SEM. n.s. not significant. Source data are provided as a Source Data file.
