Journal: Nature Communications

Article Title: Synaptic proteome diversity is shaped by the levels of glutamate receptors and their regulatory proteins

doi: 10.1038/s41467-025-65490-9

Figure Lengend Snippet: a Workflow and bioinformatic tools used to establish the proteome of synapses from the trisynaptic loop. b . Overlap between proteins from the trisynaptic loop and two reference proteomes. Proteome I, this study. Proteome II, PSDII from Distler et al. . c Sunburst plot of SynGO Cellular Component terms enriched among synaptic proteins from the trisynaptic loop. d –f Relative abundances of GluA2 (d, left), Vamp1 ( d , right), Shisa6 ( e , left), Prkar2a ( e , right), mGluR2 ( f , left) and Ptprd ( f , right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mea n ± SEM. Statistical test, one-way ANOVA, post-hoc Fisher’s LSD test, sample size (n, biological replicates) for GluA2, 8; Vamp1, 8; Shisa6, 8; Prkar2a, 8; mGluR2, 12 and Ptprd 16. * p < 0.05, *** p < 0.001, **** p < 0.0001. Grm2 dimer band, the most abundant form, was analyzed. g –l Left: Representative double immunofluorescences of the postsynaptic marker Psd95 (in red) with Homer2 ( g ), Calcineurin (Ppp3ca, h ), Homer3 ( i ), Band 4.1-like protein 1 (Epb41l1, k ) and Mpp2 ( l ) and the presynaptic marker vGlut1 (in red) with Synaptoporin (Synpr, j ). Right: Quantification of overlapping signal (see also Suppl. Figs. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± SEM. Statistical test, one-way ANOVA, post-hoc Fisher’s LSD test, sample size (n, biological replicates) for Homer2, 10; Ppp3ca, 22; Homer3, 22; Synpr, 9; Epb41l1, 10 and Mpp2, 20. **** p < 0.0001. m–o Percentages of differentially expressed (DE) proteins in CA3–CA1 ( m ), DG–CA3 ( n ), and EC–DG ( o ) synapses with concordant or discordant in situ hybridization (ISH) levels. p–r DE proteins with increased scRNA-seq levels: CA3–CA1 synapses, in CA3do or CA1do neurons ( p , blue); DG–CA3 synapses, in DG or CA3do neurons ( q , green); EC–DG synapses, in EC or DG neurons ( r , orange). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used: Psd95 (#3450; Cell Signaling, [RRID:AB_2292883]); Synaptophysin (Ab8049; Abcam [SY38], [RRID:AB_2198854]); GluA2 (MAB397; Millipore [RRID:AB_2113875]; Shisa6 (NBP2-85726; Novus Biologicals [RRID:AB_3427376]); mGluR2 (# 191 103; Synaptic Systems [RRID:AB_2232859]; Prkar2a (ab32514; Abcam [RRID:AB_777289]); Ptprd (NBP2-94767; Novus Biologicals [RRID:AB_3464681]) and Vamp1 (#13151; Cell Signaling [RRID:AB_2798132]).

Techniques: Western Blot, Marker, In Situ Hybridization

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 1. Ptprd+/ and Ptprd/ Mice Show Deficits in Proliferation and Enhancement of Differentiation of Cultured Cortical NPCs (A) RT-PCR for Ptprd mRNA in the E11–E18 cortex. b-Actin mRNA was used as a loading control. (B) qRT-PCR for Ptprd mRNA in the E12–E18 cortex. Data are expressed as fold over E12 cortex. (C) Western blot of PTPRD in total cortical lysates from E13 to E16. The blot was reprobed for ERK1/2 as a loading control. (D) Image of E13.5 Ptprd+/+ (PTPRD-WT [wild type], left panel) and Ptprd/ (PTPRD-KO [knockout], right panel) cortical sections immunostained for PTPRD (red). The cortical plate (CP), subventricular zone (SVZ), ventricular zone (VZ), and ventricle (V) are denoted. The tissue was counterstained with Hoechst (blue) to show nuclei. The inset shows the magnified image of the boxed area. Scale bar, 200 mm. (E) Images of E12.5 cortical NPCs cultured for 3 days and then immunostained for PTPRD (green), bIII-tubulin (red), and Sox2 (magenta). Arrows and arrowheads indicate PTPRD+ neurons and cortical NPCs, respectively. Scale bar, 50 mm. (F–M) E12.5 cortical NPCs from single PTPRD-WT, Ptprd+/ (PTPRD-HET [heterozygous]), or PTPRD-KO embryos were cultured and analyzed 3 days later. (F–H) Cells were immunostained for Sox2 (red) and bIII-tubulin (magenta) after 3 days (F) and the proportions of Sox2+ (G) or bIII-tubulin+ (H) cells were deter- mined. Scale bar, 50 mm. **p < 0.01, ***p < 0.001; n = 3 embryos per genotype. (I–K) Cells were immunostained for Ki67 (green) and Sox2 (red) after 3 days (I), and the proportion of total Ki67+ cells was determined (J). Alternatively, the proportion of total Ki67+;Sox2+/+ cells over the total Sox2+ cells was determined (K). (L and M) Cells immunostained for cleaved caspase-3 and Sox2 or bIII-tubulin (not shown) after 3 days and the CC3+ cells proportion over total Sox2+ cells (L) and total bIII-tubulin+ (M) was determined. Scale bar, 50 mm. **p < 0.01, ***p < 0.001; n = 3 embryos per genotype.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER murine shRNA TrkB-specific sequence: 50-GCTCCTTAAG GATAACGAA-30 This paper N/A murine Mek1/2 shRNA-specific sequence: 50-GCTGATC CACCTGGAGATCAA-30 Kevin Janes Lab Addgene # 72570 Recombinant DNA pCMV-mPTPRD-Myc/DDK Origene Cat#: MR227256 pCMV-hPTPRD-Myc/DDK Origene Cat#: RC214115 murine PTPRD cDNA ORF clone Origene Cat#: MG226685 pEF-DEST51 Invitrogen Cat#: 12285011 pSUPER.retro.Neo+GFP Oligoengine Cat#: VEC-PRT-0005/0006 pENTR-GST6P-1 Eric Campeau Lab Addgene #17741 pLKO.3G Christophe Benoist & Diane Mathis Lab Addgene # 14748 pCLX-UBI-VenusN Patrick Salmon Lab Addgene # 27247 pCMV6-mMek-Myc-Flag Origene Cat#: PS100016 Software and Algorithms CellProfiler software Carpenter et al., 2006 https://cellprofiler.org/; RRID:SCR_007358 Prism 8 GraphPad https://www.graphpad.com/; RRID:SCR_002798 Adobe Illustrator CC Adobe https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 Adobe Photoshop CC Adobe https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 ZEN software Zeiss https://www.zeiss.com/microscopy/int/products/ microscope-software/zen.html; RRID: SCR_013672 LAS X software Leica https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/; RRID: SCR_013673

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR, Western Blot, Knock-Out

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 2. Ptprd Knockdown Decreases Proliferation and Increases Differentiation, While PTPRD Overexpression Increases Proliferation and Decreases Differentiation of Cultured Embryonic Cortical NPCs (A) Western blot for PTPRD in lysates of HEK293 cells transfected with a murine Ptprd expression vector together with control or Ptprd shRNAs or with an empty shRNA vector. The blot was reprobed for ERK1/2. (B) qRT-PCR for Ptprd mRNA in HEK293 cells transfected with a murine Ptprd expression vector together with control or Ptprd shRNA. ***p < 0.001. Data are representative of three independent experiments. (C) Cortical NPCs with decreased PTPRD expression due to knockdown. E12.5 cortical NPCs were co-transfected with cytoplasmic EGFP and control or Ptprd shRNA and then immunostained for EGFP and PTPRD (transfected cells are indicated with arrows and non-transfected cells with arrowheads). (D–G) E12.5 cortical NPCs were co-transfected with nuclear EGFP and control or Ptprd shRNA and then analyzed 3 days later. (D and E) Cultures were immunostained for EGFP (green, D) and Ki67 (red, D; double-labeled cells are indicated with arrows) and the proportion of total EGFP+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER murine shRNA TrkB-specific sequence: 50-GCTCCTTAAG GATAACGAA-30 This paper N/A murine Mek1/2 shRNA-specific sequence: 50-GCTGATC CACCTGGAGATCAA-30 Kevin Janes Lab Addgene # 72570 Recombinant DNA pCMV-mPTPRD-Myc/DDK Origene Cat#: MR227256 pCMV-hPTPRD-Myc/DDK Origene Cat#: RC214115 murine PTPRD cDNA ORF clone Origene Cat#: MG226685 pEF-DEST51 Invitrogen Cat#: 12285011 pSUPER.retro.Neo+GFP Oligoengine Cat#: VEC-PRT-0005/0006 pENTR-GST6P-1 Eric Campeau Lab Addgene #17741 pLKO.3G Christophe Benoist & Diane Mathis Lab Addgene # 14748 pCLX-UBI-VenusN Patrick Salmon Lab Addgene # 27247 pCMV6-mMek-Myc-Flag Origene Cat#: PS100016 Software and Algorithms CellProfiler software Carpenter et al., 2006 https://cellprofiler.org/; RRID:SCR_007358 Prism 8 GraphPad https://www.graphpad.com/; RRID:SCR_002798 Adobe Illustrator CC Adobe https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 Adobe Photoshop CC Adobe https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 ZEN software Zeiss https://www.zeiss.com/microscopy/int/products/ microscope-software/zen.html; RRID: SCR_013672 LAS X software Leica https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/; RRID: SCR_013673

Techniques: Knockdown, Over Expression, Cell Culture, Western Blot, Transfection, Expressing, Plasmid Preparation, Control, shRNA, Quantitative RT-PCR, Labeling

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 3. PTPRD-HET and PTPRD-KO Embryos Display Deficits in Cortical Precursor Proliferation, Neuronal Numbers, and Neuronal Localization (A–N) Pregnant mothers were injected with BrdU at E13.5 and 1 (A–E), 24 (F–K), or 48 h later (L–Q; see Figure S2), cortices were removed from their E13.5, E14.5, or E15.5 PTPRD-WT, PTPRD-HET (not shown), and PTPRD-KO progeny, respectively. Coronal cortical sections were then immunostained for BrdU (green; A, C, F, H, and J; see Figure S2), the radial precursor marker Pax6 (red; A, F, and L), the intermediate progenitor marker Tbr2 (red; C, H, and M), or the deep cortical layer marker Tbr1 (red; E, J, and N). These sections were then quantified for the proportion of BrdU+ cells that were also positive for Pax6 (B and G), Tbr2 (D and I), or Tbr1 (K). (O–Q). Cortical sections as in (A) –(N) were immunostained, and a cortical column of defined width spanning the lateral ventricles to the meninges (see Figure S2) was quantified for the total numbers of cells expressing Pax6 (O), Tbr2 (P), or Tbr1 (Q). *p < 0.05; **p < 0.01; ***p < 0.001; n = 3 embryos per genotype. The CP and VZ are denoted. In all of the images, scale bars represent 50 mm. In all of the panels, the error bars denote SEMs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER murine shRNA TrkB-specific sequence: 50-GCTCCTTAAG GATAACGAA-30 This paper N/A murine Mek1/2 shRNA-specific sequence: 50-GCTGATC CACCTGGAGATCAA-30 Kevin Janes Lab Addgene # 72570 Recombinant DNA pCMV-mPTPRD-Myc/DDK Origene Cat#: MR227256 pCMV-hPTPRD-Myc/DDK Origene Cat#: RC214115 murine PTPRD cDNA ORF clone Origene Cat#: MG226685 pEF-DEST51 Invitrogen Cat#: 12285011 pSUPER.retro.Neo+GFP Oligoengine Cat#: VEC-PRT-0005/0006 pENTR-GST6P-1 Eric Campeau Lab Addgene #17741 pLKO.3G Christophe Benoist & Diane Mathis Lab Addgene # 14748 pCLX-UBI-VenusN Patrick Salmon Lab Addgene # 27247 pCMV6-mMek-Myc-Flag Origene Cat#: PS100016 Software and Algorithms CellProfiler software Carpenter et al., 2006 https://cellprofiler.org/; RRID:SCR_007358 Prism 8 GraphPad https://www.graphpad.com/; RRID:SCR_002798 Adobe Illustrator CC Adobe https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 Adobe Photoshop CC Adobe https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 ZEN software Zeiss https://www.zeiss.com/microscopy/int/products/ microscope-software/zen.html; RRID: SCR_013672 LAS X software Leica https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/; RRID: SCR_013673

Techniques: Injection, Marker, Expressing

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 4. Acute Ptprd Knockdown In- creases Intermediate Progenitors and Neu- rons In Vivo (A–H) E13/E14 cortices were electroporated with EGFP and control or Ptprd shRNA, and coronal cortical sections were immunostained 3 days later for EGFP (green) and Pax6 (red; C), Tbr2 (red; E), or NeuN (red; G) and quantified for the relative loca- tion of EGFP+ cells in the different cortical regions (B) or for the proportions of EGFP+ cells expressing Pax6 (D), Tbr2 (F), or NeuN (H). The image showed in (A) was generated by stitching. *p < 0.05; **p < 0.01; ***p < 0.001. In all cases, data are representative of three inde- pendent experiments. The boxed regions in (C) and (E) are shown at higher magnification as insets, with arrows indicating double-positive cells. The CP, VZ, SVZ, and intermediate zone (IZ) are indi- cated. Scale bar, 50 mm. In all of the panels, the error bars denote SEMs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER murine shRNA TrkB-specific sequence: 50-GCTCCTTAAG GATAACGAA-30 This paper N/A murine Mek1/2 shRNA-specific sequence: 50-GCTGATC CACCTGGAGATCAA-30 Kevin Janes Lab Addgene # 72570 Recombinant DNA pCMV-mPTPRD-Myc/DDK Origene Cat#: MR227256 pCMV-hPTPRD-Myc/DDK Origene Cat#: RC214115 murine PTPRD cDNA ORF clone Origene Cat#: MG226685 pEF-DEST51 Invitrogen Cat#: 12285011 pSUPER.retro.Neo+GFP Oligoengine Cat#: VEC-PRT-0005/0006 pENTR-GST6P-1 Eric Campeau Lab Addgene #17741 pLKO.3G Christophe Benoist & Diane Mathis Lab Addgene # 14748 pCLX-UBI-VenusN Patrick Salmon Lab Addgene # 27247 pCMV6-mMek-Myc-Flag Origene Cat#: PS100016 Software and Algorithms CellProfiler software Carpenter et al., 2006 https://cellprofiler.org/; RRID:SCR_007358 Prism 8 GraphPad https://www.graphpad.com/; RRID:SCR_002798 Adobe Illustrator CC Adobe https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 Adobe Photoshop CC Adobe https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 ZEN software Zeiss https://www.zeiss.com/microscopy/int/products/ microscope-software/zen.html; RRID: SCR_013672 LAS X software Leica https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/; RRID: SCR_013673

Techniques: Knockdown, In Vivo, Control, shRNA, Expressing, Generated

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 5. PTPRD-HET and PTPRD-KO Mice Show Increased Neuronal Number and Mis- localized Neurons (A–F) E18.5 PTPRD-WT, PTPRD-HET (not shown), and PTPRD-KO coronal cortical sections were immunostained for Tbr1 (A; red) and Satb2 (D; red; sections were counterstained with Hoechst 33258 in blue) and quantified for relative total number of Tbr1+ cells (B), and for the relative number of Satb2+ cells (E). Also, the relative location of Tbr1+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER murine shRNA TrkB-specific sequence: 50-GCTCCTTAAG GATAACGAA-30 This paper N/A murine Mek1/2 shRNA-specific sequence: 50-GCTGATC CACCTGGAGATCAA-30 Kevin Janes Lab Addgene # 72570 Recombinant DNA pCMV-mPTPRD-Myc/DDK Origene Cat#: MR227256 pCMV-hPTPRD-Myc/DDK Origene Cat#: RC214115 murine PTPRD cDNA ORF clone Origene Cat#: MG226685 pEF-DEST51 Invitrogen Cat#: 12285011 pSUPER.retro.Neo+GFP Oligoengine Cat#: VEC-PRT-0005/0006 pENTR-GST6P-1 Eric Campeau Lab Addgene #17741 pLKO.3G Christophe Benoist & Diane Mathis Lab Addgene # 14748 pCLX-UBI-VenusN Patrick Salmon Lab Addgene # 27247 pCMV6-mMek-Myc-Flag Origene Cat#: PS100016 Software and Algorithms CellProfiler software Carpenter et al., 2006 https://cellprofiler.org/; RRID:SCR_007358 Prism 8 GraphPad https://www.graphpad.com/; RRID:SCR_002798 Adobe Illustrator CC Adobe https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 Adobe Photoshop CC Adobe https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 ZEN software Zeiss https://www.zeiss.com/microscopy/int/products/ microscope-software/zen.html; RRID: SCR_013672 LAS X software Leica https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/; RRID: SCR_013673

Techniques:

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 6. PTPRD Knockdown Increases the Activity of the Neurogenic RTKs PDGFR and TrkB and Its Downstream Substrate Erk1/2 (A) Schematic representation of PTPRD protein structure and mutant PTPRD without catalytic domain (PTPRDDCD) constructs. (B–D) E12.5 cortical precursor cells were co-transfected with nuclear EGFP and control or Ptprd shRNA plus or minus an expression construct encoding PTPRDDCD. The expression of the construct was detected by western blots using an antibody against PTPRD (B). Cultures were immunostained 3 days later, and the proportion of EGFP+ cells that were also positive for Ki67 (C) or bIII-tubulin (D) was determined. **p < 0.01; ***p < 0.001. Data are representative of three independent experiments. (E) Schematic representation of PTPRD substrate-trapping constructs; GST-tagged PTPRD (GST-PTPRD-WT) or PTPRD mutated to generate a substrate- trapping protein (GST-PTPRD-CS or GST-PTPRD-DA). (F) E12.5 cortical lysates were incubated with GST-PTPRD-WT or PTPRD mutated GST-PTPRD-CS or GST-PTPRD-DA. The GST-tagged PTPRD and associated proteins were then pulled down, and the complexes were analyzed by western blots, probed with anti-PDGFRb, anti-TrkB, or, as a control, anti-GST. (G and H) Neurospheres were generated from the cortex of E14 PTPRD-WT, PTPRD-HET, and PTPRD-KO mice and analyzed by western blot, probing with antibodies against phospho-PDGFRb (tyrosine 1,009), phospho-TrkB (Tyr 512), phospho-MEK, or phospho-ERK. Blots were then reprobed with antibodies for the detection of total PDGFRb, total TrkB, total MEK, or total ERK as loading controls.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER murine shRNA TrkB-specific sequence: 50-GCTCCTTAAG GATAACGAA-30 This paper N/A murine Mek1/2 shRNA-specific sequence: 50-GCTGATC CACCTGGAGATCAA-30 Kevin Janes Lab Addgene # 72570 Recombinant DNA pCMV-mPTPRD-Myc/DDK Origene Cat#: MR227256 pCMV-hPTPRD-Myc/DDK Origene Cat#: RC214115 murine PTPRD cDNA ORF clone Origene Cat#: MG226685 pEF-DEST51 Invitrogen Cat#: 12285011 pSUPER.retro.Neo+GFP Oligoengine Cat#: VEC-PRT-0005/0006 pENTR-GST6P-1 Eric Campeau Lab Addgene #17741 pLKO.3G Christophe Benoist & Diane Mathis Lab Addgene # 14748 pCLX-UBI-VenusN Patrick Salmon Lab Addgene # 27247 pCMV6-mMek-Myc-Flag Origene Cat#: PS100016 Software and Algorithms CellProfiler software Carpenter et al., 2006 https://cellprofiler.org/; RRID:SCR_007358 Prism 8 GraphPad https://www.graphpad.com/; RRID:SCR_002798 Adobe Illustrator CC Adobe https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 Adobe Photoshop CC Adobe https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 ZEN software Zeiss https://www.zeiss.com/microscopy/int/products/ microscope-software/zen.html; RRID: SCR_013672 LAS X software Leica https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/; RRID: SCR_013673

Techniques: Knockdown, Activity Assay, Mutagenesis, Construct, Transfection, Control, shRNA, Expressing, Western Blot, Incubation, Generated

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 7. Decreasing MEK/ERK1/2 Activity or TrkB Rescues the Perturbations in Neurogenesis Induced by Ptprd Knockdown (A) E12.5 cortical NPC cultures were treated or not treated with MEK/ERK inhibitors, trametinib, or PD98059. Western blot of phospho-MEK and phospho-ERK. Blots were then reprobed with antibodies for total MEK or total ERK as loading controls. (B and C) E12.5 cortical NPCs from single PTPRD-WT, PTPRD-HET, or PTPRD-KO embryos were treated or not treated with 100 nM trametinib (black column) or 50 mM PD98059 (gray column). Three days later, cultures were immunostained for EGFP and bIII-tubulin (B), and the proportion of transfected newborn neurons was quantified (C). Scale bar, 50 mm. *p < 0.05; **p < 0.01. n = 7 for WT experiments; n = 4 for HET and KO experiments. (D and E) E12.5 cortical NPCs were co-transfected with nuclear EGFP and control or Ptprd shRNA and treated or not treated with 100 nM trametinib (D) or 50 mM PD98059 (E). Three days later, cultures were immunostained for EGFP and bIII-tubulin, and the proportion of transfected newborn neurons was determined. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. Data are representative of three independent experiments. (F and H) Western blot for TrkB (F) or MEK1/2 (H) in lysates of HEK293 cells transfected with control or TrkB shRNA (F) or MEK shRNA (H) vector. The blot was reprobed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (G and I) E12.5 cortical NPCs were co-transfected with nuclear EGFP and control or Ptprd shRNA and co-transfected with TrkB shRNA (G) or MEK shRNA (I). Three days later, cultures were immunostained for EGFP and bIII-tubulin, and the proportion of transfected newborn neurons was determined. **p < 0.01; ***p < 0.001; ns, not significant. Data are representative of three independent experiments. Error bars denote SEMs.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER murine shRNA TrkB-specific sequence: 50-GCTCCTTAAG GATAACGAA-30 This paper N/A murine Mek1/2 shRNA-specific sequence: 50-GCTGATC CACCTGGAGATCAA-30 Kevin Janes Lab Addgene # 72570 Recombinant DNA pCMV-mPTPRD-Myc/DDK Origene Cat#: MR227256 pCMV-hPTPRD-Myc/DDK Origene Cat#: RC214115 murine PTPRD cDNA ORF clone Origene Cat#: MG226685 pEF-DEST51 Invitrogen Cat#: 12285011 pSUPER.retro.Neo+GFP Oligoengine Cat#: VEC-PRT-0005/0006 pENTR-GST6P-1 Eric Campeau Lab Addgene #17741 pLKO.3G Christophe Benoist & Diane Mathis Lab Addgene # 14748 pCLX-UBI-VenusN Patrick Salmon Lab Addgene # 27247 pCMV6-mMek-Myc-Flag Origene Cat#: PS100016 Software and Algorithms CellProfiler software Carpenter et al., 2006 https://cellprofiler.org/; RRID:SCR_007358 Prism 8 GraphPad https://www.graphpad.com/; RRID:SCR_002798 Adobe Illustrator CC Adobe https://www.adobe.com/products/illustrator.html; RRID: SCR_010279 Adobe Photoshop CC Adobe https://www.adobe.com/products/photoshop.html; RRID: SCR_014199 ZEN software Zeiss https://www.zeiss.com/microscopy/int/products/ microscope-software/zen.html; RRID: SCR_013672 LAS X software Leica https://www.leica-microsystems.com/products/ microscope-software/p/leica-las-x-ls/; RRID: SCR_013673

Techniques: Activity Assay, Knockdown, Western Blot, Transfection, Control, shRNA, Plasmid Preparation

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 2. Ptprd Knockdown Decreases Proliferation and Increases Differentiation, While PTPRD Overexpression Increases Proliferation and Decreases Differentiation of Cultured Embryonic Cortical NPCs (A) Western blot for PTPRD in lysates of HEK293 cells transfected with a murine Ptprd expression vector together with control or Ptprd shRNAs or with an empty shRNA vector. The blot was reprobed for ERK1/2. (B) qRT-PCR for Ptprd mRNA in HEK293 cells transfected with a murine Ptprd expression vector together with control or Ptprd shRNA. ***p < 0.001. Data are representative of three independent experiments. (C) Cortical NPCs with decreased PTPRD expression due to knockdown. E12.5 cortical NPCs were co-transfected with cytoplasmic EGFP and control or Ptprd shRNA and then immunostained for EGFP and PTPRD (transfected cells are indicated with arrows and non-transfected cells with arrowheads). (D–G) E12.5 cortical NPCs were co-transfected with nuclear EGFP and control or Ptprd shRNA and then analyzed 3 days later. (D and E) Cultures were immunostained for EGFP (green, D) and Ki67 (red, D; double-labeled cells are indicated with arrows) and the proportion of total EGFP+

Article Snippet: The target sequence for murine Ptprd shRNA was cloned into pSUPER.retro.Neo+GFP. cDNAs encoding murine (pCMV-m PTPRD -Myc/DDK) and human PTPRD (pCMV-hPTPRD-Myc/DDK) were purchased from Origene.

Techniques: Knockdown, Over Expression, Cell Culture, Western Blot, Transfection, Expressing, Plasmid Preparation, Control, shRNA, Quantitative RT-PCR, Labeling

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 4. Acute Ptprd Knockdown In- creases Intermediate Progenitors and Neu- rons In Vivo (A–H) E13/E14 cortices were electroporated with EGFP and control or Ptprd shRNA, and coronal cortical sections were immunostained 3 days later for EGFP (green) and Pax6 (red; C), Tbr2 (red; E), or NeuN (red; G) and quantified for the relative loca- tion of EGFP+ cells in the different cortical regions (B) or for the proportions of EGFP+ cells expressing Pax6 (D), Tbr2 (F), or NeuN (H). The image showed in (A) was generated by stitching. *p < 0.05; **p < 0.01; ***p < 0.001. In all cases, data are representative of three inde- pendent experiments. The boxed regions in (C) and (E) are shown at higher magnification as insets, with arrows indicating double-positive cells. The CP, VZ, SVZ, and intermediate zone (IZ) are indi- cated. Scale bar, 50 mm. In all of the panels, the error bars denote SEMs.

Article Snippet: The target sequence for murine Ptprd shRNA was cloned into pSUPER.retro.Neo+GFP. cDNAs encoding murine (pCMV-m PTPRD -Myc/DDK) and human PTPRD (pCMV-hPTPRD-Myc/DDK) were purchased from Origene.

Techniques: Knockdown, In Vivo, Control, shRNA, Expressing, Generated

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 6. PTPRD Knockdown Increases the Activity of the Neurogenic RTKs PDGFR and TrkB and Its Downstream Substrate Erk1/2 (A) Schematic representation of PTPRD protein structure and mutant PTPRD without catalytic domain (PTPRDDCD) constructs. (B–D) E12.5 cortical precursor cells were co-transfected with nuclear EGFP and control or Ptprd shRNA plus or minus an expression construct encoding PTPRDDCD. The expression of the construct was detected by western blots using an antibody against PTPRD (B). Cultures were immunostained 3 days later, and the proportion of EGFP+ cells that were also positive for Ki67 (C) or bIII-tubulin (D) was determined. **p < 0.01; ***p < 0.001. Data are representative of three independent experiments. (E) Schematic representation of PTPRD substrate-trapping constructs; GST-tagged PTPRD (GST-PTPRD-WT) or PTPRD mutated to generate a substrate- trapping protein (GST-PTPRD-CS or GST-PTPRD-DA). (F) E12.5 cortical lysates were incubated with GST-PTPRD-WT or PTPRD mutated GST-PTPRD-CS or GST-PTPRD-DA. The GST-tagged PTPRD and associated proteins were then pulled down, and the complexes were analyzed by western blots, probed with anti-PDGFRb, anti-TrkB, or, as a control, anti-GST. (G and H) Neurospheres were generated from the cortex of E14 PTPRD-WT, PTPRD-HET, and PTPRD-KO mice and analyzed by western blot, probing with antibodies against phospho-PDGFRb (tyrosine 1,009), phospho-TrkB (Tyr 512), phospho-MEK, or phospho-ERK. Blots were then reprobed with antibodies for the detection of total PDGFRb, total TrkB, total MEK, or total ERK as loading controls.

Article Snippet: The target sequence for murine Ptprd shRNA was cloned into pSUPER.retro.Neo+GFP. cDNAs encoding murine (pCMV-m PTPRD -Myc/DDK) and human PTPRD (pCMV-hPTPRD-Myc/DDK) were purchased from Origene.

Techniques: Knockdown, Activity Assay, Mutagenesis, Construct, Transfection, Control, shRNA, Expressing, Western Blot, Incubation, Generated

Journal: Cell reports

Article Title: The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis.

doi: 10.1016/j.celrep.2019.11.033

Figure Lengend Snippet: Figure 7. Decreasing MEK/ERK1/2 Activity or TrkB Rescues the Perturbations in Neurogenesis Induced by Ptprd Knockdown (A) E12.5 cortical NPC cultures were treated or not treated with MEK/ERK inhibitors, trametinib, or PD98059. Western blot of phospho-MEK and phospho-ERK. Blots were then reprobed with antibodies for total MEK or total ERK as loading controls. (B and C) E12.5 cortical NPCs from single PTPRD-WT, PTPRD-HET, or PTPRD-KO embryos were treated or not treated with 100 nM trametinib (black column) or 50 mM PD98059 (gray column). Three days later, cultures were immunostained for EGFP and bIII-tubulin (B), and the proportion of transfected newborn neurons was quantified (C). Scale bar, 50 mm. *p < 0.05; **p < 0.01. n = 7 for WT experiments; n = 4 for HET and KO experiments. (D and E) E12.5 cortical NPCs were co-transfected with nuclear EGFP and control or Ptprd shRNA and treated or not treated with 100 nM trametinib (D) or 50 mM PD98059 (E). Three days later, cultures were immunostained for EGFP and bIII-tubulin, and the proportion of transfected newborn neurons was determined. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. Data are representative of three independent experiments. (F and H) Western blot for TrkB (F) or MEK1/2 (H) in lysates of HEK293 cells transfected with control or TrkB shRNA (F) or MEK shRNA (H) vector. The blot was reprobed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (G and I) E12.5 cortical NPCs were co-transfected with nuclear EGFP and control or Ptprd shRNA and co-transfected with TrkB shRNA (G) or MEK shRNA (I). Three days later, cultures were immunostained for EGFP and bIII-tubulin, and the proportion of transfected newborn neurons was determined. **p < 0.01; ***p < 0.001; ns, not significant. Data are representative of three independent experiments. Error bars denote SEMs.

Article Snippet: The target sequence for murine Ptprd shRNA was cloned into pSUPER.retro.Neo+GFP. cDNAs encoding murine (pCMV-m PTPRD -Myc/DDK) and human PTPRD (pCMV-hPTPRD-Myc/DDK) were purchased from Origene.

Techniques: Activity Assay, Knockdown, Western Blot, Transfection, Control, shRNA, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Chondroitinase and Antidepressants Promote Plasticity by Releasing TRKB from Dephosphorylating Control of PTPσ in Parvalbumin Neurons

doi: 10.1523/jneurosci.2228-20.2020

Figure Lengend Snippet: Fig. 2: Deletion of CSPG receptor PTPσ facilitates TRKB phosphorylation and delays closure of 577

Article Snippet: MG87.TRKA and MG87.TRKB cells were 138 transfected with PTPσ (RefSeq number NM_019140) Myc-DDK-tagged open-reading frame (ORF) 139 plasmid purchased from OriGene (#RR209636), pCMV6-Entry vector with C-terminal Myc-DDK Tag 140 (#PS100001) was used to transfect control cells.

Techniques: Phospho-proteomics