Journal: Cells

Article Title: Caffeine Mitigates Adenosine-Mediated Angiogenic Properties of Choroidal Endothelial Cells Through Antagonism of A 1 Adenosine Receptor and PI3K-AKT Axis

doi: 10.3390/cells15010087

Figure Lengend Snippet: The effects of AR antagonists on ChEC migration and AKT activation. ( A ) Cell migration was assessed using a Transwell assay in the presence of the vehicle (con), NECA (10 µM), or a combination of NECA with caffeine (400 µM), DPCPX (A 1 AR antagonist, 100 nM), Istradefylline (A 2A AR antagonist, 1 µM), MRS1754 (A 2B AR antagonist, 100 nM), or MRS1523 (A 3 AR antagonist, 100 nM). Migration was quantified by counting the number of cells that migrated through the membrane in 8 high-power fields (×200; *** p < 0.001, * p < 0.05, n = 3). ( B ) AKT activation was determined by Western blot analysis using AKT phosphorylation-specific and total protein antibodies after the incubation of ChEC with the vehicle (con), NECA (10 µM), and combinations of NECA with caffeine, DPCPX (A 1 AR antagonist, 100 nM), or Istradefylline (A 2A AR antagonist, 1 µM) for 1 h. ( C ) Band intensities were assessed using the ImageJ software.

Article Snippet: Caffeine citrate (51754-0500-1; Exela Pharma Sciences, Lenoir, NC, USA), Bz-ATP (2′(3′)-O-(4-benzoylbenzoyl) adenosine -5ʹ -triphosphate, tri(triethylammonium) salt (HY-136254), NECA (5′-N-ethylcarboxamidoadenosine, HY-103173), DPCPX (A 1 AR antagonist, HY-100937), Istradefylline (A 2A AR antagonist, HY-10888), MRS1754 (A 2B AR antagonist, HY-14121), MRS1523 (A 3 AR antagonist, HY-121119), SU5402 (VEGFR2 inhibitor, HY-10407) (Med Chem Express, Monmouth Junction, NJ, USA), LY294002 (PI3K inhibitor, L9908; Sigma), and AZ 11645373 (P2X7 receptor antagonist, 3317; R&D Systems, Minneapolis, MN, USA) were prepared with the desired concentration in EC growth medium.

Techniques: Migration, Activation Assay, Transwell Assay, Membrane, Western Blot, Phospho-proteomics, Incubation, Software

Journal: Cells

Article Title: Caffeine Mitigates Adenosine-Mediated Angiogenic Properties of Choroidal Endothelial Cells Through Antagonism of A 1 Adenosine Receptor and PI3K-AKT Axis

doi: 10.3390/cells15010087

Figure Lengend Snippet: Effects of caffeine and A 1 , A 2A , A 2B , and A 3 AR antagonism on intracellular cAMP production in ChEC. Intracellular cAMP levels were assessed following treatment with the vehicle (con), NECA (10 µM), or a combination of NECA with caffeine (400 µM), DPCPX (100 nM; A 1 AR antagonist), Istradefylline (A 2A AR antagonist; 1 µM), MRS1754 (A 2B AR antagonist, 100 nM), or MRS1523 (A 3 AR antagonist, 100 nM). Antagonists were pre-treated for 10 min, followed by 10 min incubation with NECA (* p < 0.05, ** p < 0.01; n = 3).

Article Snippet: Caffeine citrate (51754-0500-1; Exela Pharma Sciences, Lenoir, NC, USA), Bz-ATP (2′(3′)-O-(4-benzoylbenzoyl) adenosine -5ʹ -triphosphate, tri(triethylammonium) salt (HY-136254), NECA (5′-N-ethylcarboxamidoadenosine, HY-103173), DPCPX (A 1 AR antagonist, HY-100937), Istradefylline (A 2A AR antagonist, HY-10888), MRS1754 (A 2B AR antagonist, HY-14121), MRS1523 (A 3 AR antagonist, HY-121119), SU5402 (VEGFR2 inhibitor, HY-10407) (Med Chem Express, Monmouth Junction, NJ, USA), LY294002 (PI3K inhibitor, L9908; Sigma), and AZ 11645373 (P2X7 receptor antagonist, 3317; R&D Systems, Minneapolis, MN, USA) were prepared with the desired concentration in EC growth medium.

Techniques: Incubation

Journal: Regenerative Therapy

Article Title: Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis

doi: 10.1016/j.reth.2024.01.006

Figure Lengend Snippet: Isolation of bMSCs-derived exosomes . (A) The size of bMSCs-derived vesicles detected by NanoSight. (B) The level of exosomal markers CD63 and CD81 in bMSCs-derived vesicles detected by WB.

Article Snippet: After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively.

Techniques: Isolation, Derivative Assay

Journal: Scientific reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis.

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: Figure 5. Slit3 down-regulates cell surface Robo4 and induces Robo4 internalization. (A) GW but not Slit3 reduced Cdh5 shedding. dECs in serum-free cultured were supplemented with BSA or Slit3 with or without GW, the duo-inhibitor for ADAM10 and ADAM. The conditioned media and cell lysate were probed with an anti-Cdh5 antibody in Western blot. (B) Slit3 reduced cell surface Robo4, and this was blocked by EIPA. Starved dECs were treated with Slit3 or BSA in the absence or presence of EIPA, and then cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. (C) Slit3 induced cell surface Robo4 internalization, and this was blocked by EIPA. After biotinylation of cell surface proteins at 4 °C, the hRobo4-HA-FLAG expressing dECs were treated with Slit3 or BSA in the absence or presence of EIPA at 37 °C. Cells were lysed immediately (total biotinylated protein preserved) or after MENSA treatment (MESNA cleaves uninternalized biotinylated protein), and biotinylated proteins were pulled down using streptavidin-coated beads. hRobo4-HA-FLAG were probed in western blot with anti-FLAG antibody. (D) Slit3 inhibited Robo4 shedding, and this was restored by EIPA. dECs were treated with Slit3 or BSA in the absence or presence of EIPA, and then sRobo4 in conditioned medium, and total Robo4 and actin in cell lysates were probed. sRobo4 was normalized to the total Robo4. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for a two-group comparison. ns, not significant; *p < 0.05; **p < 0.01.

Article Snippet: Histagged recombinant mouse Slit3 (3629-SL-050) was from R&D Systems, PMA (ab120297) from Abcam, TAPI-2 (187034-31-7) from Cayman Chemical, GW282026X (AOB3632) from AOBIOUS Inc., Polybrene (sc-134220) from Santa Cruz Biotechnology, and GI254023x (260264-93-5), LPS (#L7770) and PEI (#764965) were from Sigma-Aldrich.

Techniques: Cell Culture, Western Blot, Flow Cytometry, Staining, Expressing, Comparison

Journal: bioRxiv

Article Title: A ternary Neurexin-T178-PTPR complex represents a core-module of neuronal synapse organization

doi: 10.1101/2024.07.16.603715

Figure Lengend Snippet: ( A ), Proteomic meAP-MS approach for quantitative analysis of NRX-associated trans-synaptic protein complexes. ( B ), MS-derived protein abundance of NRX isoforms in plasma membrane-enriched fractions from adult mouse brain. Data are mean ± SD of 4 brain samples. ( C ), t-SNE plot of target normalized ratio (tnR)-values determined for all proteins (grey dots; Methods) identified in eight APs with the four distinct anti-NRX1-3 ABs with preimmunization IgGs and antiserum as negative controls and NRX1A as a reference for normalization. Right panel, extension of framed area around the target proteins (NRX1-3) highlighting a cluster of consistently co-purified proteins of mainly pre-, post- and trans-synaptic origin as indicated by the color-code. ( D ), Protein abundance determined by MS in mouse brain membrane fractions plotted against the respective protein abundances determined by MS in anti-NRX1-3 AP eluates (normalized to the abundance of NRX1A). Proteins with tight binding to the NRX-target(s) are located on the diagonal. Data are mean ± SD of two mouse brains and two independent AP eluates each. ( E ), Table summarizing a selected set of proteins specifically co-purified in APs with the indicated isoform-specific anti-NRX antibodies. Specificity was determined with target-KO (NRX1, NRX3) and IgG controls. Note that PTPRD,S and T178B are interactors of all NRX isoforms.

Article Snippet: The following antibodies were used for APs: anti-Nrx1-3 (Millipore, #ABN161-I), anti-Nrx1-3 (AB generation by LifeTein, #RB2987, rabbit, epitope: mouse NRX3A aa 1523-1571); anti-Nrx1-3 (Synaptic Systems, #175003), anti-Nrx1-3 (AB generation by AbFrontier, #3-4-rb1, rabbit, epitope: mouse NRX3A aa 1471-1491), anti-Nrx1 (Synaptic Systems, #175103), anti-Nrx1 (RnD Systems, #AF4524), anti-Nrx2 (AB generation by LifeTein, #RB4227, rabbit, epitope: mouse NRX2A aa 266-282), anti-Nrx2 (AB generation by LifeTein, #RB4226, rabbit, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (AB generation by LifeTein, rabbit, #RB4225, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (Abcam, #ab34245), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-1-rb1, epitope: mouse NRX3A aa 1349-1368), anti-Nrx3 (Synaptic Systems, #175303), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb1, epitope: mouse NRX3A aa 1377-1397), anti-Nrx3 (RnD Systems, #AF5269), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb2, epitope: mouse NRX3A aa 1377-1397), anti-T178B (AB generation by LifeTein, rabbit, #RB4235, epitope: mouse T178B aa 273-294), anti-T178B (AB generation by AmsBio, rabbit, #RB3967, epitope: mouse T178B aa 273-294), anti-HA (Roche, #11867423001), anti-HA (Thermo Fisher, #26183), anti-PTPRS (RnD Systems, #AF3430), anti-PTPRS (MediMabs, #MM-0020-P), anti-PTPRS (Invitrogen, #PA5-47487), anti-CAH10 (RnD Systems, #MAB2189), anti-CAH10 (raised against recombinant CAH10 ( , rabbit, #Hadlai), anti-CAH10 (raised against recombinant CAH10 , rabbit, #Habeba), anti-CAC1A (FrontierInstitute, #GP-Af810), anti-CAC1E (Alomone, #ACC-006), anti-TAFA1 (RnD Systems, #AF5154), anti-TAFA2 (RnD Systems, #AF4179), anti-NXPH1 (RnD Systems, #AF4208), anti-NXPH3 (RnD Systems, #AF5098), anti-LRRT4 (Neuromab, #75-261), anti-LRRT4 (AbFrontier, rabbit, #C1, raised against mouse LRRT4 aa 548-568), anti-LRRT4 (RnD Systems, #AF5377), anti-A4 (Abgent, #ABIN1741750), anti-A4 (Sigma, #A8717), anti-GBRA1 (Synaptic Systems, #224203), anti-GBRG (Synaptic Systems, #224003), anti-RTN4R (RnD Systems, #AF1440), anti-LRRT1 (MerckMillipore, #ABN642), anti-LRRT2 (NeuroMab, #75-364), anti-GRM4 (Abcam, #ab53088), anti-GRM4 (Abcam, #ab184302), anti-GRM4 (Bioworld, #BS1144), anti-NLGN2 (Synaptic Systems, #129511), anti-NLGN3 (NeuroMab, #75-158), anti-Syt11 (Synaptic Systems, #270003), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3961, epitope: mouse XKR6 aa 85-102), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3962, epitope: mouse XKR6 aa 85-102), anti-XKR6 (Signalway Antibody, #42864), IgG (Millipore, #12-370), rabbit pre-immune-sera (LifeTein).

Techniques: Derivative Assay, Membrane, Purification, Binding Assay

Journal: bioRxiv

Article Title: A ternary Neurexin-T178-PTPR complex represents a core-module of neuronal synapse organization

doi: 10.1101/2024.07.16.603715

Figure Lengend Snippet: Relative abundance of NRX isoforms (alpha, beta, gamma) was determined by quantitative MS in un-solubilized membrane fractions prepared from adult mouse brains. Proteins were separated by denaturing SDS-PAGE, the gel was cut at 45 and 140 kDa and protein abundance was determined in individual sections. Sequence information unique for NRX1ψ were exclusively found in lower molecular weight (MW) sections, peptides unique for NRX1² were identified between 45-140 kDa. All peptides matching the NRX1ψ sequence in the 0-45 kDa sections were used to calculate the abundance of this NRX proteoform. Similarly, protein abundances of NRX1² and NRX1α were determined from the middle and high MW sections, respectively.

Article Snippet: The following antibodies were used for APs: anti-Nrx1-3 (Millipore, #ABN161-I), anti-Nrx1-3 (AB generation by LifeTein, #RB2987, rabbit, epitope: mouse NRX3A aa 1523-1571); anti-Nrx1-3 (Synaptic Systems, #175003), anti-Nrx1-3 (AB generation by AbFrontier, #3-4-rb1, rabbit, epitope: mouse NRX3A aa 1471-1491), anti-Nrx1 (Synaptic Systems, #175103), anti-Nrx1 (RnD Systems, #AF4524), anti-Nrx2 (AB generation by LifeTein, #RB4227, rabbit, epitope: mouse NRX2A aa 266-282), anti-Nrx2 (AB generation by LifeTein, #RB4226, rabbit, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (AB generation by LifeTein, rabbit, #RB4225, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (Abcam, #ab34245), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-1-rb1, epitope: mouse NRX3A aa 1349-1368), anti-Nrx3 (Synaptic Systems, #175303), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb1, epitope: mouse NRX3A aa 1377-1397), anti-Nrx3 (RnD Systems, #AF5269), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb2, epitope: mouse NRX3A aa 1377-1397), anti-T178B (AB generation by LifeTein, rabbit, #RB4235, epitope: mouse T178B aa 273-294), anti-T178B (AB generation by AmsBio, rabbit, #RB3967, epitope: mouse T178B aa 273-294), anti-HA (Roche, #11867423001), anti-HA (Thermo Fisher, #26183), anti-PTPRS (RnD Systems, #AF3430), anti-PTPRS (MediMabs, #MM-0020-P), anti-PTPRS (Invitrogen, #PA5-47487), anti-CAH10 (RnD Systems, #MAB2189), anti-CAH10 (raised against recombinant CAH10 ( , rabbit, #Hadlai), anti-CAH10 (raised against recombinant CAH10 , rabbit, #Habeba), anti-CAC1A (FrontierInstitute, #GP-Af810), anti-CAC1E (Alomone, #ACC-006), anti-TAFA1 (RnD Systems, #AF5154), anti-TAFA2 (RnD Systems, #AF4179), anti-NXPH1 (RnD Systems, #AF4208), anti-NXPH3 (RnD Systems, #AF5098), anti-LRRT4 (Neuromab, #75-261), anti-LRRT4 (AbFrontier, rabbit, #C1, raised against mouse LRRT4 aa 548-568), anti-LRRT4 (RnD Systems, #AF5377), anti-A4 (Abgent, #ABIN1741750), anti-A4 (Sigma, #A8717), anti-GBRA1 (Synaptic Systems, #224203), anti-GBRG (Synaptic Systems, #224003), anti-RTN4R (RnD Systems, #AF1440), anti-LRRT1 (MerckMillipore, #ABN642), anti-LRRT2 (NeuroMab, #75-364), anti-GRM4 (Abcam, #ab53088), anti-GRM4 (Abcam, #ab184302), anti-GRM4 (Bioworld, #BS1144), anti-NLGN2 (Synaptic Systems, #129511), anti-NLGN3 (NeuroMab, #75-158), anti-Syt11 (Synaptic Systems, #270003), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3961, epitope: mouse XKR6 aa 85-102), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3962, epitope: mouse XKR6 aa 85-102), anti-XKR6 (Signalway Antibody, #42864), IgG (Millipore, #12-370), rabbit pre-immune-sera (LifeTein).

Techniques: Membrane, SDS Page, Sequencing, Molecular Weight

Journal: bioRxiv

Article Title: A ternary Neurexin-T178-PTPR complex represents a core-module of neuronal synapse organization

doi: 10.1101/2024.07.16.603715

Figure Lengend Snippet: Target-normalized ratio (tnR)-values of selected proteins specifically (co-)purified in 12 anti-NRX1-3 APs with four different anti-NRX1-3 ABs (see also ). tnR-values >0.25 indicate specific co-purification (with the target). Average indicates average-tnRs calculated from all MS-identified proteins in the respective AP-eluate, grey area represents SDs of these average-tnRs.

Article Snippet: The following antibodies were used for APs: anti-Nrx1-3 (Millipore, #ABN161-I), anti-Nrx1-3 (AB generation by LifeTein, #RB2987, rabbit, epitope: mouse NRX3A aa 1523-1571); anti-Nrx1-3 (Synaptic Systems, #175003), anti-Nrx1-3 (AB generation by AbFrontier, #3-4-rb1, rabbit, epitope: mouse NRX3A aa 1471-1491), anti-Nrx1 (Synaptic Systems, #175103), anti-Nrx1 (RnD Systems, #AF4524), anti-Nrx2 (AB generation by LifeTein, #RB4227, rabbit, epitope: mouse NRX2A aa 266-282), anti-Nrx2 (AB generation by LifeTein, #RB4226, rabbit, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (AB generation by LifeTein, rabbit, #RB4225, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (Abcam, #ab34245), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-1-rb1, epitope: mouse NRX3A aa 1349-1368), anti-Nrx3 (Synaptic Systems, #175303), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb1, epitope: mouse NRX3A aa 1377-1397), anti-Nrx3 (RnD Systems, #AF5269), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb2, epitope: mouse NRX3A aa 1377-1397), anti-T178B (AB generation by LifeTein, rabbit, #RB4235, epitope: mouse T178B aa 273-294), anti-T178B (AB generation by AmsBio, rabbit, #RB3967, epitope: mouse T178B aa 273-294), anti-HA (Roche, #11867423001), anti-HA (Thermo Fisher, #26183), anti-PTPRS (RnD Systems, #AF3430), anti-PTPRS (MediMabs, #MM-0020-P), anti-PTPRS (Invitrogen, #PA5-47487), anti-CAH10 (RnD Systems, #MAB2189), anti-CAH10 (raised against recombinant CAH10 ( , rabbit, #Hadlai), anti-CAH10 (raised against recombinant CAH10 , rabbit, #Habeba), anti-CAC1A (FrontierInstitute, #GP-Af810), anti-CAC1E (Alomone, #ACC-006), anti-TAFA1 (RnD Systems, #AF5154), anti-TAFA2 (RnD Systems, #AF4179), anti-NXPH1 (RnD Systems, #AF4208), anti-NXPH3 (RnD Systems, #AF5098), anti-LRRT4 (Neuromab, #75-261), anti-LRRT4 (AbFrontier, rabbit, #C1, raised against mouse LRRT4 aa 548-568), anti-LRRT4 (RnD Systems, #AF5377), anti-A4 (Abgent, #ABIN1741750), anti-A4 (Sigma, #A8717), anti-GBRA1 (Synaptic Systems, #224203), anti-GBRG (Synaptic Systems, #224003), anti-RTN4R (RnD Systems, #AF1440), anti-LRRT1 (MerckMillipore, #ABN642), anti-LRRT2 (NeuroMab, #75-364), anti-GRM4 (Abcam, #ab53088), anti-GRM4 (Abcam, #ab184302), anti-GRM4 (Bioworld, #BS1144), anti-NLGN2 (Synaptic Systems, #129511), anti-NLGN3 (NeuroMab, #75-158), anti-Syt11 (Synaptic Systems, #270003), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3961, epitope: mouse XKR6 aa 85-102), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3962, epitope: mouse XKR6 aa 85-102), anti-XKR6 (Signalway Antibody, #42864), IgG (Millipore, #12-370), rabbit pre-immune-sera (LifeTein).

Techniques: Purification, Copurification

Journal: bioRxiv

Article Title: A ternary Neurexin-T178-PTPR complex represents a core-module of neuronal synapse organization

doi: 10.1101/2024.07.16.603715

Figure Lengend Snippet: ( A ), Native gel separation (BN-PAGE) of protein complexes in CL47-solubilized membrane fractions from adult mouse brain Western-probed for the indicated proteins. Note close co-migration of NRX1-3A, PTPRD/S and T178B as tripartite complex with an apparent molecular weight of approximately 400 kDa. Fainter signals extending to an apparent molecular mass of 1.2 MDa reflect their integration in protein networks. ( B ), tnR-plot of APs with antibodies targeting T178B and PTPRS indicate robust mutual co-purification and enrichment of NRX1-3 and a variety of pre- and post-synaptic interactors. tnR-values were determined with IgGs as a reference. Color-code of subcellular localization is indicated. ( C ), Abundance ratio determined for proteins affinity-purified in anti-HA APs from HA-Neurexin3 AS5 (GPI-anchored splice variant of NRX3, inset) mice and in anti-NRX3 APs from wild-type mice. Note that GPI-anchored NRX3 complexes lack association with presynaptic PTPRD,S, T178B and XKR6, whereas binding of extracellular ligands was maintained. Experiments were done with two distinct HA and two NRX3 ABs, values are mean of two independent experiments. ( D ), Abundance ratio for a subset of proteins affinity-purified with anti-NRX1-3 from CL91-solubilized brain membrane fractions with and without enzymatic treatment with a mixture of heparinases (I, II, III); inset: Western-probed gel-separation indicating heparinase-mediated reduction in size of NRX3. Enzymatic activity strongly reduced co-purification of postsynaptic proteins (LRRTs, AMPAR subunits GRIA1, 2), while binding to presynaptic and extracellular/secreted interactors remained unchanged. Data are mean ± SEM of 3 experiments. ( E ), Reconstitution of ternary complexes assembled from PTPRS, NRX1A and T178B in cultured tsA cells. Specific co-purification of T178B and NRX1A with V5-tagged PTPRS in anti-V5 APs. The presence of T178B appears critical for stable complex formation with NRX1A and impacts maturation of the P-subunit of PTPRS. ( F ) Left panel, Structure of the membrane-close region of the core-module predicted by AlphaFold2-Multimer with contact sites in the transmembrane plane. Input used for AlphaFold was NRX1ψ (mouse, aa 1-139, full length), PTPRS (mouse, aa 1251-1360), T178B (human, 1-294, full length). Right panel, Scheme of the ternary complex with multiple sites/domains for protein-protein interactions on both the extracellular and the cytoplasmic side of the membrane.

Article Snippet: The following antibodies were used for APs: anti-Nrx1-3 (Millipore, #ABN161-I), anti-Nrx1-3 (AB generation by LifeTein, #RB2987, rabbit, epitope: mouse NRX3A aa 1523-1571); anti-Nrx1-3 (Synaptic Systems, #175003), anti-Nrx1-3 (AB generation by AbFrontier, #3-4-rb1, rabbit, epitope: mouse NRX3A aa 1471-1491), anti-Nrx1 (Synaptic Systems, #175103), anti-Nrx1 (RnD Systems, #AF4524), anti-Nrx2 (AB generation by LifeTein, #RB4227, rabbit, epitope: mouse NRX2A aa 266-282), anti-Nrx2 (AB generation by LifeTein, #RB4226, rabbit, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (AB generation by LifeTein, rabbit, #RB4225, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (Abcam, #ab34245), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-1-rb1, epitope: mouse NRX3A aa 1349-1368), anti-Nrx3 (Synaptic Systems, #175303), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb1, epitope: mouse NRX3A aa 1377-1397), anti-Nrx3 (RnD Systems, #AF5269), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb2, epitope: mouse NRX3A aa 1377-1397), anti-T178B (AB generation by LifeTein, rabbit, #RB4235, epitope: mouse T178B aa 273-294), anti-T178B (AB generation by AmsBio, rabbit, #RB3967, epitope: mouse T178B aa 273-294), anti-HA (Roche, #11867423001), anti-HA (Thermo Fisher, #26183), anti-PTPRS (RnD Systems, #AF3430), anti-PTPRS (MediMabs, #MM-0020-P), anti-PTPRS (Invitrogen, #PA5-47487), anti-CAH10 (RnD Systems, #MAB2189), anti-CAH10 (raised against recombinant CAH10 ( , rabbit, #Hadlai), anti-CAH10 (raised against recombinant CAH10 , rabbit, #Habeba), anti-CAC1A (FrontierInstitute, #GP-Af810), anti-CAC1E (Alomone, #ACC-006), anti-TAFA1 (RnD Systems, #AF5154), anti-TAFA2 (RnD Systems, #AF4179), anti-NXPH1 (RnD Systems, #AF4208), anti-NXPH3 (RnD Systems, #AF5098), anti-LRRT4 (Neuromab, #75-261), anti-LRRT4 (AbFrontier, rabbit, #C1, raised against mouse LRRT4 aa 548-568), anti-LRRT4 (RnD Systems, #AF5377), anti-A4 (Abgent, #ABIN1741750), anti-A4 (Sigma, #A8717), anti-GBRA1 (Synaptic Systems, #224203), anti-GBRG (Synaptic Systems, #224003), anti-RTN4R (RnD Systems, #AF1440), anti-LRRT1 (MerckMillipore, #ABN642), anti-LRRT2 (NeuroMab, #75-364), anti-GRM4 (Abcam, #ab53088), anti-GRM4 (Abcam, #ab184302), anti-GRM4 (Bioworld, #BS1144), anti-NLGN2 (Synaptic Systems, #129511), anti-NLGN3 (NeuroMab, #75-158), anti-Syt11 (Synaptic Systems, #270003), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3961, epitope: mouse XKR6 aa 85-102), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3962, epitope: mouse XKR6 aa 85-102), anti-XKR6 (Signalway Antibody, #42864), IgG (Millipore, #12-370), rabbit pre-immune-sera (LifeTein).

Techniques: Membrane, Western Blot, Migration, Molecular Weight, Copurification, Affinity Purification, Variant Assay, Binding Assay, Activity Assay, Cell Culture

Journal: bioRxiv

Article Title: A ternary Neurexin-T178-PTPR complex represents a core-module of neuronal synapse organization

doi: 10.1101/2024.07.16.603715

Figure Lengend Snippet: Western-probed SDS-PAGE separations of input and eluates of APs with anti-V5 ABs from tsA201 cells transiently expressing V5-tagged PTPRs, FLAG-tagged T178B and either NRX3A or a splice variant of NRX3A lacking the transmembrane domain and attached to the membrane via a GPI-anchor. Note that the presence of T178B appears critical for stable complex formation with NRX3A (similar as NRX1A, ). In contrast, binding of NRX3A-GPI to PTPRS is weak and independent of the presence of T178B. (B) Details to structural predictions by AlphaFold Prediction of 3D-structural arrangement of the membrane-close region of the core-module composed of NRX1ψ (mouse, aa 1-139, full length), PTPRS (mouse, aa 1251-1360) and T178B (human, 1-294, full length) by AlphaFold2-Multimer. Predicted aligned errors (PAE) provide estimates for the confidence of the correct position of two residues in the structure. Note high-confidence levels (colored blue) were only observed in the transmembrane regions; lower confidence levels are shown in yellow.

Article Snippet: The following antibodies were used for APs: anti-Nrx1-3 (Millipore, #ABN161-I), anti-Nrx1-3 (AB generation by LifeTein, #RB2987, rabbit, epitope: mouse NRX3A aa 1523-1571); anti-Nrx1-3 (Synaptic Systems, #175003), anti-Nrx1-3 (AB generation by AbFrontier, #3-4-rb1, rabbit, epitope: mouse NRX3A aa 1471-1491), anti-Nrx1 (Synaptic Systems, #175103), anti-Nrx1 (RnD Systems, #AF4524), anti-Nrx2 (AB generation by LifeTein, #RB4227, rabbit, epitope: mouse NRX2A aa 266-282), anti-Nrx2 (AB generation by LifeTein, #RB4226, rabbit, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (AB generation by LifeTein, rabbit, #RB4225, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (Abcam, #ab34245), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-1-rb1, epitope: mouse NRX3A aa 1349-1368), anti-Nrx3 (Synaptic Systems, #175303), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb1, epitope: mouse NRX3A aa 1377-1397), anti-Nrx3 (RnD Systems, #AF5269), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb2, epitope: mouse NRX3A aa 1377-1397), anti-T178B (AB generation by LifeTein, rabbit, #RB4235, epitope: mouse T178B aa 273-294), anti-T178B (AB generation by AmsBio, rabbit, #RB3967, epitope: mouse T178B aa 273-294), anti-HA (Roche, #11867423001), anti-HA (Thermo Fisher, #26183), anti-PTPRS (RnD Systems, #AF3430), anti-PTPRS (MediMabs, #MM-0020-P), anti-PTPRS (Invitrogen, #PA5-47487), anti-CAH10 (RnD Systems, #MAB2189), anti-CAH10 (raised against recombinant CAH10 ( , rabbit, #Hadlai), anti-CAH10 (raised against recombinant CAH10 , rabbit, #Habeba), anti-CAC1A (FrontierInstitute, #GP-Af810), anti-CAC1E (Alomone, #ACC-006), anti-TAFA1 (RnD Systems, #AF5154), anti-TAFA2 (RnD Systems, #AF4179), anti-NXPH1 (RnD Systems, #AF4208), anti-NXPH3 (RnD Systems, #AF5098), anti-LRRT4 (Neuromab, #75-261), anti-LRRT4 (AbFrontier, rabbit, #C1, raised against mouse LRRT4 aa 548-568), anti-LRRT4 (RnD Systems, #AF5377), anti-A4 (Abgent, #ABIN1741750), anti-A4 (Sigma, #A8717), anti-GBRA1 (Synaptic Systems, #224203), anti-GBRG (Synaptic Systems, #224003), anti-RTN4R (RnD Systems, #AF1440), anti-LRRT1 (MerckMillipore, #ABN642), anti-LRRT2 (NeuroMab, #75-364), anti-GRM4 (Abcam, #ab53088), anti-GRM4 (Abcam, #ab184302), anti-GRM4 (Bioworld, #BS1144), anti-NLGN2 (Synaptic Systems, #129511), anti-NLGN3 (NeuroMab, #75-158), anti-Syt11 (Synaptic Systems, #270003), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3961, epitope: mouse XKR6 aa 85-102), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3962, epitope: mouse XKR6 aa 85-102), anti-XKR6 (Signalway Antibody, #42864), IgG (Millipore, #12-370), rabbit pre-immune-sera (LifeTein).

Techniques: Western Blot, SDS Page, Expressing, Variant Assay, Membrane, Binding Assay

Journal: bioRxiv

Article Title: A ternary Neurexin-T178-PTPR complex represents a core-module of neuronal synapse organization

doi: 10.1101/2024.07.16.603715

Figure Lengend Snippet: ( A ), Abundance-mass profiles obtained for the indicated proteins by csBN-MS in CL-47 solubilized ER-enriched membrane fractions from adult mouse brain. Inset: Scheme of csBN-MS approach used ( ; ). Note close co-migration of NRX1A, PTPRD,S and T178A, B indicating their co-assembly into complexes of variable apparent mass. Values of protein abundance indicate close to stoichiometric association (see text). ( B ), Ratio of protein abundances determined by quantitative MS in membranes from cultured cortical neurons infected either with AAV driving sh-T178B expression or with control virus. Note profound alteration in the expression of distinct synaptic proteins by sh-RNA-mediated knock-down of T178B: Expression of PTPRD, S was decreased by ∼40%, while essential subunits of NMDA receptors (GluN2B), Na/K-ATPase (AT1B1) and Calnexin (CALX) remained unchanged. Values are mean ± SEM of three samples of cultured neurons. Inset: Western-probed gel-separations indicating the effect of AAV:sh-T178B infection. ( C ), Alterations in PTPR and NRX complexes after knockdown of T178B. Upper panel, Input and eluates of APs with anti-PTPRS and anti-NRX1-3 APs from CL91-solubilized membrane fractions of cultured neurons transduced with two different AAV-sh-T178B viruses or control virus (WT) Western-probed with antibodies specific for NRX1-3, PTPRS,D or T178B. Lower panel, Abundance ratios determined for proteins specifically purified (and co-purified) in three anti-PTPRS and anti-NRX1-3 APs from WT and AAV-sh-T178B-2 transduced cultures by quantitative MS-analysis. Data are mean ± SD of three experiments. ( D ), Immunostaining of PTPRS in hippocampal neurons shows pronounced reduction of PTPRS expression (total and on surface). Values are mean ± SEM of 12-18 neurons of four independent cultures. ( E ), Alterations in pre-synaptic function induced by AAV-sh-T178B in hippocampal neurons of adult mice four weeks after stereotactic injection (inset, upper left). Upper right panel, Bar graph summarizing the amplitudes of AMPAR-mediated sEPSCs and representative EPSC traces measured in whole-cell recordings in CA1 pyramidal cells (CA1 PCs) in the un-injected hemisphere (control, WT, black) and in infected (green) and non-infected (blue) PCs of the injected hemisphere (inset, lower left). Data are mean ± SEM of 19 (WT), 13 (infected) and 12 (non-infected) PCs. Lower panel, Bar graphs summarizing paired-pulse (PP) ratios determined in CA1 PCs with stimulation frequencies of 10 and 50 Hz and representative AMPAR-mediated current traces recorded at 10 Hz, color-coding of expression manipulation as in the upper panel. Data are mean ± SEM of 11 (WT), 6 (infected) and 8 (non-infected) PCs. Note the marked increase in PP-ratio of AMPAR-currents triggered by sh-T178B infected pre-synapses in both infected and non-infected CA1 PCs.

Article Snippet: The following antibodies were used for APs: anti-Nrx1-3 (Millipore, #ABN161-I), anti-Nrx1-3 (AB generation by LifeTein, #RB2987, rabbit, epitope: mouse NRX3A aa 1523-1571); anti-Nrx1-3 (Synaptic Systems, #175003), anti-Nrx1-3 (AB generation by AbFrontier, #3-4-rb1, rabbit, epitope: mouse NRX3A aa 1471-1491), anti-Nrx1 (Synaptic Systems, #175103), anti-Nrx1 (RnD Systems, #AF4524), anti-Nrx2 (AB generation by LifeTein, #RB4227, rabbit, epitope: mouse NRX2A aa 266-282), anti-Nrx2 (AB generation by LifeTein, #RB4226, rabbit, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (AB generation by LifeTein, rabbit, #RB4225, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (Abcam, #ab34245), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-1-rb1, epitope: mouse NRX3A aa 1349-1368), anti-Nrx3 (Synaptic Systems, #175303), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb1, epitope: mouse NRX3A aa 1377-1397), anti-Nrx3 (RnD Systems, #AF5269), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb2, epitope: mouse NRX3A aa 1377-1397), anti-T178B (AB generation by LifeTein, rabbit, #RB4235, epitope: mouse T178B aa 273-294), anti-T178B (AB generation by AmsBio, rabbit, #RB3967, epitope: mouse T178B aa 273-294), anti-HA (Roche, #11867423001), anti-HA (Thermo Fisher, #26183), anti-PTPRS (RnD Systems, #AF3430), anti-PTPRS (MediMabs, #MM-0020-P), anti-PTPRS (Invitrogen, #PA5-47487), anti-CAH10 (RnD Systems, #MAB2189), anti-CAH10 (raised against recombinant CAH10 ( , rabbit, #Hadlai), anti-CAH10 (raised against recombinant CAH10 , rabbit, #Habeba), anti-CAC1A (FrontierInstitute, #GP-Af810), anti-CAC1E (Alomone, #ACC-006), anti-TAFA1 (RnD Systems, #AF5154), anti-TAFA2 (RnD Systems, #AF4179), anti-NXPH1 (RnD Systems, #AF4208), anti-NXPH3 (RnD Systems, #AF5098), anti-LRRT4 (Neuromab, #75-261), anti-LRRT4 (AbFrontier, rabbit, #C1, raised against mouse LRRT4 aa 548-568), anti-LRRT4 (RnD Systems, #AF5377), anti-A4 (Abgent, #ABIN1741750), anti-A4 (Sigma, #A8717), anti-GBRA1 (Synaptic Systems, #224203), anti-GBRG (Synaptic Systems, #224003), anti-RTN4R (RnD Systems, #AF1440), anti-LRRT1 (MerckMillipore, #ABN642), anti-LRRT2 (NeuroMab, #75-364), anti-GRM4 (Abcam, #ab53088), anti-GRM4 (Abcam, #ab184302), anti-GRM4 (Bioworld, #BS1144), anti-NLGN2 (Synaptic Systems, #129511), anti-NLGN3 (NeuroMab, #75-158), anti-Syt11 (Synaptic Systems, #270003), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3961, epitope: mouse XKR6 aa 85-102), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3962, epitope: mouse XKR6 aa 85-102), anti-XKR6 (Signalway Antibody, #42864), IgG (Millipore, #12-370), rabbit pre-immune-sera (LifeTein).

Techniques: Membrane, Migration, Cell Culture, Infection, Expressing, Control, Virus, Knockdown, Western Blot, Transduction, Purification, Immunostaining, Injection

Journal: bioRxiv

Article Title: A ternary Neurexin-T178-PTPR complex represents a core-module of neuronal synapse organization

doi: 10.1101/2024.07.16.603715

Figure Lengend Snippet: UMAP (Uniform Manifold Approximation and Projection, ) cluster-analysis performed with the MS-derived abundance values of all proteins (total of 172) that were specifically co-purified in NRX1-3 and reverse APs with a total of 30 different target proteins. UMAP-derived subclusters (of similar behavior over the entire set of APs) were encircled with selected constituents annotated.

Article Snippet: The following antibodies were used for APs: anti-Nrx1-3 (Millipore, #ABN161-I), anti-Nrx1-3 (AB generation by LifeTein, #RB2987, rabbit, epitope: mouse NRX3A aa 1523-1571); anti-Nrx1-3 (Synaptic Systems, #175003), anti-Nrx1-3 (AB generation by AbFrontier, #3-4-rb1, rabbit, epitope: mouse NRX3A aa 1471-1491), anti-Nrx1 (Synaptic Systems, #175103), anti-Nrx1 (RnD Systems, #AF4524), anti-Nrx2 (AB generation by LifeTein, #RB4227, rabbit, epitope: mouse NRX2A aa 266-282), anti-Nrx2 (AB generation by LifeTein, #RB4226, rabbit, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (AB generation by LifeTein, rabbit, #RB4225, epitope: mouse NRX2A aa 1459-1476), anti-Nrx2 (Abcam, #ab34245), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-1-rb1, epitope: mouse NRX3A aa 1349-1368), anti-Nrx3 (Synaptic Systems, #175303), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb1, epitope: mouse NRX3A aa 1377-1397), anti-Nrx3 (RnD Systems, #AF5269), anti-Nrx3 (AB generation by AbFrontier, rabbit, #3-2-rb2, epitope: mouse NRX3A aa 1377-1397), anti-T178B (AB generation by LifeTein, rabbit, #RB4235, epitope: mouse T178B aa 273-294), anti-T178B (AB generation by AmsBio, rabbit, #RB3967, epitope: mouse T178B aa 273-294), anti-HA (Roche, #11867423001), anti-HA (Thermo Fisher, #26183), anti-PTPRS (RnD Systems, #AF3430), anti-PTPRS (MediMabs, #MM-0020-P), anti-PTPRS (Invitrogen, #PA5-47487), anti-CAH10 (RnD Systems, #MAB2189), anti-CAH10 (raised against recombinant CAH10 ( , rabbit, #Hadlai), anti-CAH10 (raised against recombinant CAH10 , rabbit, #Habeba), anti-CAC1A (FrontierInstitute, #GP-Af810), anti-CAC1E (Alomone, #ACC-006), anti-TAFA1 (RnD Systems, #AF5154), anti-TAFA2 (RnD Systems, #AF4179), anti-NXPH1 (RnD Systems, #AF4208), anti-NXPH3 (RnD Systems, #AF5098), anti-LRRT4 (Neuromab, #75-261), anti-LRRT4 (AbFrontier, rabbit, #C1, raised against mouse LRRT4 aa 548-568), anti-LRRT4 (RnD Systems, #AF5377), anti-A4 (Abgent, #ABIN1741750), anti-A4 (Sigma, #A8717), anti-GBRA1 (Synaptic Systems, #224203), anti-GBRG (Synaptic Systems, #224003), anti-RTN4R (RnD Systems, #AF1440), anti-LRRT1 (MerckMillipore, #ABN642), anti-LRRT2 (NeuroMab, #75-364), anti-GRM4 (Abcam, #ab53088), anti-GRM4 (Abcam, #ab184302), anti-GRM4 (Bioworld, #BS1144), anti-NLGN2 (Synaptic Systems, #129511), anti-NLGN3 (NeuroMab, #75-158), anti-Syt11 (Synaptic Systems, #270003), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3961, epitope: mouse XKR6 aa 85-102), anti-XKR6 (AB generation by AmsBio, rabbit, #RB3962, epitope: mouse XKR6 aa 85-102), anti-XKR6 (Signalway Antibody, #42864), IgG (Millipore, #12-370), rabbit pre-immune-sera (LifeTein).

Techniques: Derivative Assay, Purification