Journal: The Journal of Neuroscience
Article Title: LAR-RPTPs Directly Interact with Neurexins to Coordinate Bidirectional Assembly of Molecular Machineries
doi: 10.1523/JNEUROSCI.1091-20.2020
Figure Lengend Snippet: Rescue experiments using lentiviruses expressing target shRNA-resistant proteins show specificity in the target shRNA-derived synaptogenic effects driven by Slitrk6 or Nlgn1 in heterologous synapse formation analyses. A, Representative images of heterologous synapse-formation activities of Nlgn1 and Slitrk6. Cultured hippocampal neurons were infected with the indicated protein KD lentiviruses and/or the target-specific rescue expression lentiviruses at DIV4 and cocultured with Nlgn1- or Slitrk6-expressing HEK293T cells for 12 h (Nlgn1; DIV11-12) or 24 h (Slitrk6; DIV12). Hemi-synapse induction was analyzed by double-immunostaining for HA/EGFP (blue) and VGLUT1 (red). Scale bar, 10 μm. CSK1, Caskin-1; RBP2, RIM-BP2. For expression levels of the shRNA-resistant vectors used in Figure 5, see Extended Data Figure 5-1. B, Quantification of synapse-formation activity in A by measuring the ratio of red staining intensity to blue staining intensity. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ANOVA with a nonparametric Kruskal–Wallis test. n indicates the number of cells as follows: sh-Control/Slitrk6/VGLUT1, n = 26; sh-MIM-B/Slitrk6/VGLUT1, n = 14; + MIM-B (res)/Slitrk6/VGLUT1, n = 12; sh-Caskins/Slitrk6/VGLUT1, n = 17; + CSK1 (res)/Slitrk6/VGLUT1, n = 12; sh-Abl/Slitrk6/VGLUT1, n = 14; + Abl1 (res)/Slitrk6/VGLUT1, n = 16; sh-Ena/Slitrk6/VGLUT1, n = 14; + Ena (res)/Slitrk6/VGLUT1, n = 13; sh-ELKS/Slitrk6/VGLUT1, n = 13; + ELKS2 (res)/Slitrk6/VGLUT1, n = 19; sh-SYD1A/Slitrk6/VGLUT1, n = 16; + SYD1A (res)/Slitrk6/VGLUT1, n = 11; sh-CASK/Slitrk6/VGLUT1, n = 13; + CASK (res)/Slitrk6/VGLUT1, n = 15; sh-RIM-BPs/Slitrk6/VGLUT1, n = 15; + RBP2 (res)/Slitrk6/VGLUT1, n = 14; sh-Control/Nlgn1/VGLUT1, n = 23; sh-MIM-B/Nlgn1/VGLUT1, n = 13; + MIM-B (res)/Nlgn1/VGLUT1, n = 19; sh-Caskins/Nlgn1/VGLUT1, n = 20; + CSK1 (res)/Nlgn1/VGLUT1, n = 17; sh-Abl/Nlgn1/VGLUT1, n = 15; + Abl1 (res)/Nlgn1/VGLUT1, n = 19; sh-Ena/Nlgn1/VGLUT1, n = 17; + Ena (res)/Nlgn1/VGLUT1, n = 18; sh-ELKS/Nlgn1/VGLUT1, n = 16; + ELKS2 (res)/Nlgn1/VGLUT1, n = 15; sh-SYD1A/Nlgn1/VGLUT1, n = 14; + SYD1A (res)/Nlgn1/VGLUT1, n = 23; sh-CASK/Nlgn1/VGLUT1, n = 18; + CASK (res)/Nlgn1/VGLUT1, n = 20; sh-RIM-BPs/Nlgn1/VGLUT1, n = 19; and + RBP2 (res)/Nlgn1/VGLUT1, n = 15. p values for each comparison are as follows: sh-Control versus sh-MIM-B/Slitrk6/VGLUT1, p = 0.9366; sh-Control versus + MIM-B (res)/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus sh-Caskins/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus + CSK1 (res)/Slitrk6/VGLUT1, p = 0.7744; sh-Control versus sh-Abl/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus + Abl1 (res)/Slitrk6/VGLUT1, p = 0.8745; sh-Control versus sh-Ena/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus + Ena (res)/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus sh-ELKS/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus + ELKS2 (res)/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus sh-SYD1A/Slitrk6/VGLUT1, p = 0.0195; sh-Control versus + SYD1A (res)/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus sh-CASK/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus + CASK (res)/Slitrk6/VGLUT1, p = 0.6365; sh-Control versus sh-RIM-BPs/Slitrk6/VGLUT1, p = 0.0006; sh-Control versus + RBP2 (res)/Slitrk6/VGLUT1, p > 0.9999; sh-Control versus sh-MIM-B/Nlgn1/VGLUT1, p = 0.0006; sh-Control versus + MIM-B (res)/Nlgn1/VGLUT1, p = 0.8354; sh-Control versus sh-Caskins/Nlgn1/VGLUT1, p = 0.6949; sh-Control versus + CSK1 (res)/Nlgn1/VGLUT1, p > 0.9999; sh-Control versus sh-Abl/Nlgn1/VGLUT1, p = 0.0006; sh-Control versus + Abl1 (res)/Nlgn1/VGLUT1, p = 0.3353; sh-Control versus sh-Ena/Nlgn1/VGLUT1, p = 0.0001; sh-Control versus + Ena (res)/Nlgn1/VGLUT1, p > 0.9999; sh-Control versus sh-ELKS/Nlgn1/VGLUT1, p > 0.9999; sh-Control versus + ELKS2 (res)/Nlgn1/VGLUT1, p = 0.9148; sh-Control versus sh-SYD1A/Nlgn1/VGLUT1, p = 0.0060; sh-Control versus + SYD1A (res)/Nlgn1/VGLUT1, p > 0.9999; sh-CASK/Nlgn1/VGLUT1, p = 0.3146; sh-Control versus + CASK (res)/Nlgn1/VGLUT1, p = 0.0628; sh-Control versus sh-RIM-BPs/Nlgn1/VGLUT1, p = 0.0013; and sh-Control versus + RBP2 (res)/Nlgn1/VGLUT1, p > 0.9999. CSK1, Caskin-1; RBP2, RIM-BP2.
Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-HA (clone 16B12; BioLegend, RRID: AB_2565006 ); rabbit polyclonal anti-HA (Sigma, RRID: AB_260070 ); mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-VGLUT1 (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-PTPσ (clone 17G7.2; MediMabs; RRID: AB_1808357 ); rabbit polyclonal anti-Nlgn2 (Synaptic Systems, RRID: AB_993011 ); rabbit monoclonal anti-TrkC (clone C44H5; Cell Signaling; RRID: AB_2155283 ); rabbit monoclonal anti-Enah (Cell Signaling; RRID: AB_1031036 ); mouse monoclonal anti-ABL (clone 8E9; BD Bioscience; RRID: AB_2220994 ); rabbit polyclonal anti-GABA A γ2 (Synaptic Systems; RRID: AB_2263066 ); rabbit polyclonal anti-RIM-BP2 (Synaptic Systems 316 103; RRID: AB_2620052 ); rabbit polyclonal anti-SYD1A (Fitzgerald; RRID: AB_10811953 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma Millipore, RRID: AB_2100013 ); rabbit polyclonal anti-Nrxn1α antibody (Millipore; RRID: AB_10917110 ); and mouse monoclonal anti-Csp2 (Developmental Studies Hybridoma Bank, RRID: AB_10805296 ).
Techniques: Expressing, shRNA, Derivative Assay, Cell Culture, Infection, Double Immunostaining, Activity Assay, Staining