(A–D) β-galactosidase staining of adult Nxph4βgeo/+ mice shows signals in mammillary body-related circuits (A), circumventricular organs (B–C), and cerebellar-vestibular circuits (D). Blue staining …
Brain regions expressing Nxph4.
(A) Targeting scheme to generate Nxph4-βgeo allele. Doted lines indicate homologous recombination sites. ES cells with correct Nxph4-βgeo allele were obtained from KOMP. (B) Genotype analysis of …
(A) Plots of weight as a function of age (male n = 13, female n = 16–18; #, difference between HET and KO; * difference between WT and KO). (B) Latency to fall from the accelerating rotarod plotted …
(A) RNA in situ hybridization with a probe against Nxph4 only detected Nxph4 signal in WT but not KO mice. Scale bars: 2 mm. (B) RT-qPCR from cerebellar samples with specific primers for Nxph4 (n = 3…
(A) A domain model of Nxph4 (adapted from Missler and Südhof, 1998). I: signal peptide; II: a variable domain; III: a conserved domain; IV: a linker region; V: C-terminal domain. Positions of …
(A) A schematic diagram of the strategy to generate an Nxph4-3xFLAG knock-in allele. An sgRNA is designed to excise the coding region corresponding to the C-terminal region of Nxph4 third domain. In …
(A) Immunoblot analysis of fractions derived from different steps of synaptosomal preparation. Synapse specific protein PSD-95 showed enriched signal in purified synaptosomes as expected. Somatic …
(A) Synaptosomes (tissue used: olfactory bulb, hypothalamus, midbrain, hindbrain, and the cerebellum) from Nxph4-3xFLAG KI or WT (negative control) mice were precipitated with an antibody against …
(A) HA-neurexin1α and Nxph4-3xFLAG were co-expressed in HEK293T cells. Cell lysates were precipitated with beads conjugated with an anti-FLAG antibody. Bound proteins as well as input were analyzed …
(A,B) Cerebellar synaptosomes from three Nxph4-3xFLAG KI or WT (negative control) mice were precipitated with an antibody against FLAG. Bound proteins as well as 2% input were analyzed by …
(A) A simplified diagram illustrating the inhibitory circuit of cerebellar granular layer and single-cell recording experimental design. GC: granule cells; GoC: Golgi Cells; pf: parallel fibers. …
(A–B) Statistical analysis of mIPSC decay (A, t-test) and rise time (B, Mann-Whitney U test). Data are presented as mean ± SEM. n.s., not significant.
(A) Sample traces of granule cell EPSC, evoked by stimulating mossy fibers, exhibited paired-pulse depression at ISI (inter-stimulus interval) 50 ms and ISI 100 ms from WT and KO mice. (B–F) Summary …
(A) Miniature IPSCs recorded from the KI and WT cerebellar granule cells. From left to right: representative mIPSC traces, mean mIPSC frequency, amplitude, rise, and decay (n = 11–14 cells; 5mice). …
(A) Gephyrin and vGAT staining in the cerebellar granular layer indicates Golgi-granule inhibitory synapses. (B) Quantification of puncta co-expressing gephyrin and vGAT as an indicator of …
(A) In the cerebellar granular layer, vGAT marks the Golgi cell inhibitory terminals and vGlut1 marks the mossy fiber endings. Scale bar: 20 μm. (B) Immunoblotting analysis of GABAARα1/6 in the …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Mouse monoclonal anti-FLAG M2 | Sigma-Aldrich | Cat# F1804 RRID:AB_262044 | (1:1000) |
Antibody | Rabbit polyclonal anti-pan Neurexin-1 | Millipore | Cat# ABN161 RRID:AB_10917110 | (1:1000) |
Antibody | Mouse monoclonal anti-HA1.1 | Bioledgend | Cat# 901513, RRID:AB_2565335 | (1:5000) |
Antibody | Mouse monoclonal anti-Vinculin | Sigma-Aldrich | Cat# V9131, RRID:AB_477629 | (1:10000) |
Antibody | Rabbit polyclonal anti-GABAARα1 | Synaptic System | Cat# 224 203, RRID:AB_2232180 | (1:1000) |
Antibody | Rabbit polyclonal anti-GABAARα6 | Synaptic System | Cat# 224 603, RRID:AB_2619945 | (1:1000) |
Antibody | Mouse monoclonal anti-Vglut1 | Millipore | Cat# MAB5502, RRID:AB_262185 | (1:1000) |
Antibody | Guinea pig polyclonal anti-vGAT | Frontier Institute co. Ltd | Cat#: VGAT-GP-Af1000, RRID: AB_2571624 | (1:1000) |
Antibody | Mouse monoclonal anti-beta Actin | Abcam | Cat#: ab20272 RRID:AB_445482 | (1:2000) |
Antibody | Rabbit polyclonal anti-TGF beta 1 | Abcam | Cat#: ab92486, RRID:AB_10562492 | (1:1000) |
Antibody | Rabbit polyclonal anti-MeCP2 | Zoghbi Lab | #0535 | (1:1000) |
Antibody | Mouse monoclonal anti-Gephyrin | Synaptic system | Cat#: 14701, RRID:AB_887717 | (1:1000) |
Antibody | Rabbit polyclonal anti- PSD-95 | Cell signaling | Cat # 2507, RRID:AB_561221 | (1:1000) |
Recombinant DNA reagent | pLenti-mCherry | Addgene | Cat# 36084, RRID:Addgene_36084 | |
Recombinant DNA reagent | pLenti-Nxph4-3xFLAG-mCherry | This paper | N/A | Results subsection‘Nxph4 is a secreted glycoprotein’ |
Recombinant DNA reagent | pLenti--Nxph4-4Q-HA-mCherry | This paper | N/A | Results subsection ‘Nxph4 is a secreted glycoprotein’ |
Recombinant DNA reagent | pLenti--Nxph4-6A-HA-mCherry | This paper | N/A | Results subsection ‘Nxph4 is a secreted glycoprotein’ |
Recombinant DNA reagent | pCMV-Nxph4-HA | This paper | N/A | Results subsection‘Nxph4 is a secreted glycoprotein’ |
Recombinant DNA reagent | pCMV-Nxph4-4Q-HA | This paper | N/A | Results subsection‘Nxph4 is a secreted glycoprotein’ |
Recombinant DNA reagent | pCMV-Nxph4-6A-HA | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
Recombinant DNA reagent | pCAG-HA-Nrxn1β S4(+) | Addgene | Cat# 59410, RRID:Addgene_59410 | |
Recombinant DNA reagent | pCAG-HA-Nrxn1β S4(-) | Addgene | Cat# 59409, RRID:Addgene_59409 | |
Recombinant DNA reagent | pCAG-HA-Nrxn1α | Addgene | Cat# 58266, RRID:Addgene_58266 | |
Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS1 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS2 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS3 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS4 | This paper | N/A | Results subsection‘Nxph4 interacts with α-neurexin in vivo’ |
Recombinant DNA reagent | pCAG-HA-Nrxn1α-LNS5 | This paper | N/A | Results subsection ‘Nxph4 interacts with α-neurexin in vivo’ |
Recombinant DNA reagent | pCMV-GABARα1N-HA | This paper | N/A | Results subsection ‘Nxph4 interacts with α-neurexin in vivo’ |
Recombinant DNA reagent | pCMV-GABARα6N-HA | This paper | N/A | Results subsection ‘Nxph4 interacts with α-neurexin in vivo’ |
Chemical compound, drug | X-Gal | Thermo Fisher | Cat# 15520034 | |
Chemical compound, drug | Lipofectamine 2000 | Thermo Fisher | Cat# 11668500 | |
Commercial assay, kit | Antigen Unmasking Solution (citrate based) | Vector Laboratories | Cat# H-3300 | |
Commercial assay, kit | Avidin/Biotin Blocking Kit | Vector Laboratories | Cat# SP-2001 | |
Commercial assay, kit | TSA Kit | Invitrogen | Cat# T-20932 | |
Commercial assay, kit | Papain Dissociation System Kit | Worthington Biochemical Corp. | Cat# LK003150 | |
Commercial assay, kit | PNGase F | NEB | Cat# P0704S | |
Commercial assay, kit | miRNeasy Mini Kit | Qiagen | Cat#: 217004 | |
Commercial assay, kit | M-MLV Reverse Transcriptase | Thermo Fisher | Cat#: 28025013 | |
Commercial assay, kit | iTaq Universal SYBR Green SuperMix | BIO-RAD | Cat#: 1725124 | |
Cell line (Homo sapiens) | HEK293T cells | ATCC | RRID:CVCL_0063 | |
Strain, strain background (Mus musculus) | Mouse: FVB | The Jackson Laboratory | Stock No: 001800 | |
Strain, strain background (Mus musculus) | Mouse: Nxph4 βgeo+/- | This paper | N/A | The Jackson Laboratory: 033791 |
Strain, strain background (Mus musculus) | Mouse: Nxph4 FLAG/FLAG | This paper | N/A | The Jackson Laboratory: 033792 |
Sequence-based reagent | Nxph4βgeo+/- genotyping forward primer: 5’-AAAGACTAGCAGACGCAGCA | This paper | N/A | Materials and methods subsection ‘Nxph4-βgeo mouse’ |
Sequence-based reagent | Nxph4βgeo+/- genotyping reverse primer: 3’-CCCTAACTCCCCCAAACAGA | This paper | N/A | Materials andmethods subsection ‘Nxph4-βgeo mouse’ |
Sequence-based reagent | Nxph4 FLAG/FLAGgenotyping forward primer: 5’-TCAAGTTCTCGCTGTTGGTG | This paper | N/A | Materials and methods subsection‘Nxph4FLAG/FLAG knock-in mouse’ |
Sequence-based reagent | Nxph4 FLAG/FLAG genotypingreverse primer: 3’-TTCCACGTGGCAATTAAAAG | This paper | N/A | Materials and methods subsection‘Nxph4FLAG/FLAG knock-in mouse’ |