Journal: Frontiers in Synaptic Neuroscience

Article Title: Interaction and Subcellular Association of PRRT1/SynDIG4 With AMPA Receptors

doi: 10.3389/fnsyn.2021.705664

Figure Lengend Snippet: PRRT1 interacts with AMPAR subunits GluA1-GluA4. (A) Co-immunoprecipitation (Co-IP) experiments were performed with anti-HA antibody on HEK293 cell lysates expressing Flag-GluA1 and HA-PRRT1. Immunoblotting (IB) of input and immunoprecipitated (IP) samples with anti-Flag (top) and anti-HA (bottom) antibodies shows co-immunoprecipitation of Flag-GluA1 with HA-PRRT1. (B) Co-IP of Flag-GluA2 with HA-PRRT1. (C) Co-IP of Flag-GluA3 with HA-PRRT1. (D) Co-IP of Flag-GluA4 with HA-PRRT1. (E) Weak co-IP of Flag-GluA1 with HA-PRRT2. (F) No co-IP of unrelated protein Flag-neurexin (Flag-Nrxn1) with HA-PRRT1.

Article Snippet: The following primary antibodies were used: anti-PRRT1 (ProteinTech, 17261-1-AP, RRID:AB_2878371 ), anti-PRRT1 (NeuroMab, 75-409, RRID:AB_2531894 ), anti-GluA1 (Abcam, ab109450, RRID:AB_10860361 ), anti-GluA1 (NeuroMab, 75-327, RRID:AB_2315840 ), anti-MAP2 (EnCor, CPCA-MAP2, RRID:AB_2138173 ), anti-VGLUT1 (Synaptic Systems, 135 303, RRID:AB_887875 ), anti-EEA1 (Cell Signaling, C45B10, RRID:AB_2096811 ), anti-Rab7 (Cell Signaling, D95F2, RRID:AB_1904103 ), anti-Flag (Sigma, F3165, RRID:AB_259529 ), anti-HA (Thermo Fisher Scientific, 71-5500, RRID:AB_2533988 ), anti-HA (Biolegend, 16B12, RRID:AB_10064068 ), anti-GFP (Thermo Fisher Scientific, A-11122, RRID:AB_221569 ), and anti-β tubulin (Millipore, 05-661, RRID:AB_309885 ).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot

Journal: Frontiers in Synaptic Neuroscience

Article Title: Interaction and Subcellular Association of PRRT1/SynDIG4 With AMPA Receptors

doi: 10.3389/fnsyn.2021.705664

Figure Lengend Snippet: Membrane topology and AMPAR-interacting domains of PRRT1. (A) Confocal images of HEK293 cells stained with anti-HA antibody for surface (top panels) or total (bottom panels) PRRT1. PRRT1-HA showed staining on the surface but not HA-PRRT1 or PRRT1-loop-HA. Note that different coverslips were used for surface and total staining. The calibration bar equals 10 μm. (B) A cartoon showing the topology of PRRT1 based on the results of surface staining in (A) . Each of the three places where HA tag is inserted in PRRT1 is depicted with a star. (C) Co-IP experiments were performed with anti-GFP antibody on HEK293 cell lysates expressing Flag-GluA1 and GFP-PRRT1 constructs. Immunoblotting (IB) of immunoprecipitated (IP) samples with anti-Flag antibody (top panel) and of input samples with anti-Flag (middle) and anti-GFP (bottom) antibodies (right). Flag-GluA1 co-immunoprecipitated with GFP-PRRT1 full-length (FL) and NΔ144 but the co-IP with PRRT1-CΔ34 and PRRT1-CΔ60 mutants was weak or absent, respectively. Cartoons of full length and deletion constructs of PRRT1 used in co-IP are shown on the left.

Article Snippet: The following primary antibodies were used: anti-PRRT1 (ProteinTech, 17261-1-AP, RRID:AB_2878371 ), anti-PRRT1 (NeuroMab, 75-409, RRID:AB_2531894 ), anti-GluA1 (Abcam, ab109450, RRID:AB_10860361 ), anti-GluA1 (NeuroMab, 75-327, RRID:AB_2315840 ), anti-MAP2 (EnCor, CPCA-MAP2, RRID:AB_2138173 ), anti-VGLUT1 (Synaptic Systems, 135 303, RRID:AB_887875 ), anti-EEA1 (Cell Signaling, C45B10, RRID:AB_2096811 ), anti-Rab7 (Cell Signaling, D95F2, RRID:AB_1904103 ), anti-Flag (Sigma, F3165, RRID:AB_259529 ), anti-HA (Thermo Fisher Scientific, 71-5500, RRID:AB_2533988 ), anti-HA (Biolegend, 16B12, RRID:AB_10064068 ), anti-GFP (Thermo Fisher Scientific, A-11122, RRID:AB_221569 ), and anti-β tubulin (Millipore, 05-661, RRID:AB_309885 ).

Techniques: Staining, Co-Immunoprecipitation Assay, Expressing, Construct, Western Blot, Immunoprecipitation

Journal: Frontiers in Synaptic Neuroscience

Article Title: Interaction and Subcellular Association of PRRT1/SynDIG4 With AMPA Receptors

doi: 10.3389/fnsyn.2021.705664

Figure Lengend Snippet: PRRT1 co-localizes with AMPARs and resides at extrasynaptic sites on endosomes. (A) Representative confocal images of dissociated hippocampal cultures stained for PRRT1, dendritic marker MAP2 and GluA1. Also shown is an overlay of the three images (merge). PRRT1 (green) co-localizes with GluA1 (red) in the dendrite (gray) at multiple puncta which appear as yellow in the overlay. White arrows in this and subsequent panels point to prominent areas of co-localization. (B) PRRT1 (green) does not co-localize well with the excitatory pre-synaptic marker VGLUT1 (red). (C) PRRT1 (green) shows robust co-localization with TfR-containing endosomes, labeled with transfected TfR-mCherry (red) as shown in left panels. Co-localization of PRRT1 (green), TfR-mCherry (red) and GluA1 (cyan) is shown in the right panels. (D) PRRT1 (green) shows partial co-localization with the early endosome marker EEA1 (red). (E) There is minimal co-localization of PRRT1 (green) with the late endosome marker Rab7 (red). (F) The left bar graph shows quantification of the percentage co-localization of synaptic and endosomal markers (X) with PRRT1. Right bar graph shows quantification of the percentage co-localization of PRRT1 with synaptic and endosomal markers (X). In all panels, bar graphs represent means ± SEM. The calibration bar equals 5 μm. (G) Immunoblots of total or surface fraction following surface biotinylation in hippocampal cultures. GluA1 and PRRT1 are present in surface fraction but not β-tubulin. (H) Immunoblots of total or surface fraction following surface biotinylation in acute hippocampal slices. Different amounts of total protein used are indicated. GluA1 and PRRT1 are present in surface fraction but not β-tubulin.

Article Snippet: The following primary antibodies were used: anti-PRRT1 (ProteinTech, 17261-1-AP, RRID:AB_2878371 ), anti-PRRT1 (NeuroMab, 75-409, RRID:AB_2531894 ), anti-GluA1 (Abcam, ab109450, RRID:AB_10860361 ), anti-GluA1 (NeuroMab, 75-327, RRID:AB_2315840 ), anti-MAP2 (EnCor, CPCA-MAP2, RRID:AB_2138173 ), anti-VGLUT1 (Synaptic Systems, 135 303, RRID:AB_887875 ), anti-EEA1 (Cell Signaling, C45B10, RRID:AB_2096811 ), anti-Rab7 (Cell Signaling, D95F2, RRID:AB_1904103 ), anti-Flag (Sigma, F3165, RRID:AB_259529 ), anti-HA (Thermo Fisher Scientific, 71-5500, RRID:AB_2533988 ), anti-HA (Biolegend, 16B12, RRID:AB_10064068 ), anti-GFP (Thermo Fisher Scientific, A-11122, RRID:AB_221569 ), and anti-β tubulin (Millipore, 05-661, RRID:AB_309885 ).

Techniques: Staining, Marker, Labeling, Transfection, Western Blot

Journal: Frontiers in Synaptic Neuroscience

Article Title: Interaction and Subcellular Association of PRRT1/SynDIG4 With AMPA Receptors

doi: 10.3389/fnsyn.2021.705664

Figure Lengend Snippet: PRRT1 shows interaction with phosphatase PP2B. (A) Co-IP experiments were performed with anti-HA antibody on HEK293 cell lysates expressing Flag-PP2B-Aα and HA-PRRT1 constructs. Immunoblotting (IB) of input and immunoprecipitated (IP) samples with anti-Flag (top) and anti-HA (bottom) antibodies shows co-IP of Flag-PP2B-Aα with HA-PRRT1. (B) Co-IP of HA-PRRT1 with Flag-PP2B-Aα using anti-Flag antibody for immunoprecipitation. (C) Co-IP of Flag-PP2B-Aβ with HA-PRRT1. (D) Weak co-IP of PSD-95-GFP with HA-PRRT1. (E) No co-IP of Flag-Hippocalcin (Hlcn) with HA-PRRT1.

Article Snippet: The following primary antibodies were used: anti-PRRT1 (ProteinTech, 17261-1-AP, RRID:AB_2878371 ), anti-PRRT1 (NeuroMab, 75-409, RRID:AB_2531894 ), anti-GluA1 (Abcam, ab109450, RRID:AB_10860361 ), anti-GluA1 (NeuroMab, 75-327, RRID:AB_2315840 ), anti-MAP2 (EnCor, CPCA-MAP2, RRID:AB_2138173 ), anti-VGLUT1 (Synaptic Systems, 135 303, RRID:AB_887875 ), anti-EEA1 (Cell Signaling, C45B10, RRID:AB_2096811 ), anti-Rab7 (Cell Signaling, D95F2, RRID:AB_1904103 ), anti-Flag (Sigma, F3165, RRID:AB_259529 ), anti-HA (Thermo Fisher Scientific, 71-5500, RRID:AB_2533988 ), anti-HA (Biolegend, 16B12, RRID:AB_10064068 ), anti-GFP (Thermo Fisher Scientific, A-11122, RRID:AB_221569 ), and anti-β tubulin (Millipore, 05-661, RRID:AB_309885 ).

Techniques: Co-Immunoprecipitation Assay, Expressing, Construct, Western Blot, Immunoprecipitation

Journal: eNeuro

Article Title: Loss of SynDIG1 Reduces Excitatory Synapse Maturation But Not Formation In Vivo

doi: 10.1523/ENEURO.0130-16.2016

Figure Lengend Snippet: Synapse composition is unaltered in SynDIG1-deficient synapses. A , Representative immunoblots of biochemical fractions isolated from WT and SynDIG1 β-gal homozygous mutant P14 mouse brain tissue showing levels of GluA1, GluA2, GluN1, GluN2B, PSD-93, PSD-95, synaptophysin (Synapto), SynDIG1, SynDIG4, and PICK-1 present in the S1-, P2-, Syn-, and PSD-enriched fractions. Loading controls are provided by β-actin and β-tubulin immunoreactivity. B–D , Graphs depict the ratio of SynDIG1 β-gal homozygous mutant protein relative to WT levels of AMPA receptor subunits ( B ), NMDA receptor subunits ( C ), and PSD-93 and PSD-95 ( D ) in the PSD-enriched fractions. Data are the average of three independent biochemical fractionation experiments; each experiment used four to six mouse brains of each genotype. Error bars represent ±SEM.

Article Snippet: Membranes were blotted with the following primary antibodies: mouse antibodies against PSD-95 [catalog #75-028, NeuroMab (RRID:AB_2292909)], synaptophysin [catalog #101011, Synaptic Systems (RRID:AB_887824)], SynDIG1 [catalog #75-251, NeuroMab (RRID:AB_10999753)], GluA2 [catalog #75-002, NeuroMab (RRID:AB_2232661)], PSD-93 [catalog #75-057, NeuroMab (RRID:AB_2277296)], β-tubulin [catalog #05-661, Millipore (RRID:AB_309885)], GluN1 [catalog #556308, BD Biosciences (RRID:AB_396353)], and Pick1 [catalog #73-040, NeuroMab (RRID:AB_10672986)]; rabbit antibodies against SynDIG4 (Anti-Prrt1, catalog #17261-1-AP, ProteinTech), β-actin [catalog #ab8224, Abcam (RRID:AB_449644)], GluA1 [catalog #AB1504, Millipore (RRID:AB_2113602)], and GluN2B (provided by J.W.H.

Techniques: Western Blot, Isolation, Mutagenesis, Fractionation