Journal: The Journal of Cell Biology

Article Title: Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

doi: 10.1083/jcb.202004164

Figure Lengend Snippet: Identification of FAM19A1-A4 as neurexin ligands. (A) Identification of FAM19A1 and FAM19A2 as endogenous neurexin ligands. Top: Schematic of proteomic screen; bottom, alignment of Mus musculus FAM19A1-A5 amino acid sequences highlighting peptides identified by Mascot [FAM19A1, green; FAM19A2, blue; underline, TAFA domain (CX 7 CCX 13 CXCX 14 CX 11 CX 4 CX 5 CX 10 C); asterisks, colons, and periods indicate fully, strongly, or weakly conserved residues, respectively; red, DS residues in FAM19A5 that replace conserved CC residues in FAM19A1-A4]. Neurexin complexes affinity purified from Nrxn1 HA/HA or WT (negative control) adult mouse brains were analyzed by LC-MS/MS. (B and C) Recombinant FAM19A1-A4, but not FAM19A5, binds to Nrxn1β. FAM19As (FAM19A1-V5 to -A5-V5) were coexpressed in HEK293T cells with either secreted WT (Nrxn1β SS4-SS5- -ECD-Myc-6xHis) or cysteine-loop deleted Nrxn1β-ECD (Nrxn1β SS4-SS5- -ECD-ΔCysL-Myc-6xHis). Nrxn1β/FAM19A complexes in the medium were analyzed by immunoprecipitation and reducing SDS-PAGE (B) or directly by non-reducing and reducing SDS-PAGE and immunoblotting (C; magenta, Myc; green, V5; white, overlap). IB, immunoblot; IP, immunoprecipitation; **, FAM19A homodimers; *, FAM19A monomers. All experiments were independently performed at least three times.

Article Snippet: The following primary antibodies were used for immunoblotting: chicken anti-c-myc (1:500; Aves Labs, ET-MY100, RRID: AB_2313514), mouse anti-V5 (1:1,000; Invitrogen, R960-25, RRID: AB_2556564), rabbit anti-V5 (1:1,000; Millipore, AB3792), rabbit anti-Strep-tag II (1:1,000; Abcam, ab76949), rabbit anti-myc (clone 71D10; 1:1,000; Cell Signaling, rabbit mAb 2278), mouse anti-HA.11 (clone 16B12; 1:1,000; BioLegend, MMS-101R, RRID: AB_256533), mouse anti-GAPDH (clone 6C5; 1 h incubation at RT; 1:5,000; Millipore, MAB374), rabbit anti-pan-Nrxn (1–2 overnight incubation at 4°C; 1:1,000; Millipore, ABN161-I), mouse anti-GABA A R β2/3 (clone 62-3G1; 1:500; NeuroMab, 75–363, RRID: AB_2315837), mouse anti-Δ-HS (3G10 epitope, clone F69-3G10; 1:1,000; Amsbio, 370260-S), and rabbit anti-synapsin (1:1,000; homemade, YZ6079).

Techniques: Affinity Purification, Negative Control, Liquid Chromatography with Mass Spectroscopy, Recombinant, Immunoprecipitation, SDS Page, Western Blot

Journal: The Journal of Cell Biology

Article Title: Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

doi: 10.1083/jcb.202004164

Figure Lengend Snippet: Biophysical definition of the Nrxn1β/FAM19A1 complex. (A and B) Nrxn1β-ECD (Nrxn1β-ECD-Myc-6xHis) was coexpressed with FAM19A1-V5 in HEK293S GnTI − cells and purified from the medium. In the final purification step, the Nrxn1β-ECD/FAM19A1 complex elutes as a single peak (A, peak 1), as shown by Coomassie-blue staining (B) and immunoblotting for Myc and V5 (A, inset), whereas excess Nrxn1β-ECD elutes as a separate peak (A, peak 2). (C and D) Final purification step for Nrxn1β-ECD-Myc-6xHis, expressed alone, from the medium of HEK293S GnTI − cells (C, elution profile; D, Coomassie-blue-stained reducing SDS-gel). (E and F) Final purification step for FAM19A1-Twin-Strep, expressed alone, from the medium of HEK293S GnTI − cells (E, elution profile with Twin-Strep immunoblot in inset; F, Coomassie-blue-stained reducing SDS-gel). See also . (G and H) Nrxn1β-ECD forms a stoichiometric 1:1 complex with FAM19A1. Analysis by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) of the purified Nrxn1β-ECD/FAM19A1 complex and of Nrxn1β-ECD (G), and of FAM19A1 (H), which enables the determination of their precise molar masses (dRI, differential refractive index; MM, molar mass; dotted lines in B, D, and F denote fractions that were combined and analyzed). (I) Molar masses of the Nrxn1β-ECD/FAM19A1 complex, Nrxn1β-ECD, and FAM19A1 as predicted (Prot pi Protein Tool; assuming all cysteines form disulfide bonds and Nrxn1β contains one Man 5 GlcNAc 2 addition) or as experimentally determined by MALS or electrospray ionization (ESI)-MS.

Article Snippet: The following primary antibodies were used for immunoblotting: chicken anti-c-myc (1:500; Aves Labs, ET-MY100, RRID: AB_2313514), mouse anti-V5 (1:1,000; Invitrogen, R960-25, RRID: AB_2556564), rabbit anti-V5 (1:1,000; Millipore, AB3792), rabbit anti-Strep-tag II (1:1,000; Abcam, ab76949), rabbit anti-myc (clone 71D10; 1:1,000; Cell Signaling, rabbit mAb 2278), mouse anti-HA.11 (clone 16B12; 1:1,000; BioLegend, MMS-101R, RRID: AB_256533), mouse anti-GAPDH (clone 6C5; 1 h incubation at RT; 1:5,000; Millipore, MAB374), rabbit anti-pan-Nrxn (1–2 overnight incubation at 4°C; 1:1,000; Millipore, ABN161-I), mouse anti-GABA A R β2/3 (clone 62-3G1; 1:500; NeuroMab, 75–363, RRID: AB_2315837), mouse anti-Δ-HS (3G10 epitope, clone F69-3G10; 1:1,000; Amsbio, 370260-S), and rabbit anti-synapsin (1:1,000; homemade, YZ6079).

Techniques: Purification, Staining, Western Blot, SDS-Gel, Size-exclusion Chromatography, Multi-Angle Light Scattering, Refractive Index

Journal: The Journal of Cell Biology

Article Title: Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

doi: 10.1083/jcb.202004164

Figure Lengend Snippet: Exogenously added FAM19A1 protein binds weakly to surface β-neurexin in contrast to coexpressed FAM19A1 protein that binds stoichiometrically, and purified recombinant FAM19A1 exists as a monomer as well as disulfide-mediated dimers. (A–D) FAM19A1-V5-6xHis was purified from the medium of transfected Expi293 cells via pull-down using HisTrap followed by size-exclusion chromatography (SEC) on a Superdex 75 column (A). Fractions corresponding to peaks were analyzed by SDS-PAGE (B–D). The peak 2 fraction was found to exist as a monomer under non-reducing conditions and was used for subsequent cell surface binding assays. **, FAM19A1 homodimers; *, FAM19A1 monomers. (E and F) HEK293T cells were transfected with constructs encoding HA-tagged Nrxn1β, Nrxn1β-ΔCysL, Nrxn1β-ΔCHO (all Ser and Thr mutated to glycines to prevent O -glycosylation and HS modification), or the PDGFR transmembrane domain (TM). 48 h later, purified recombinant FAM19A1-V5-6xHis (A–D) was added at a final concentration of 100 nM for 4 h at 4°C to prevent endocytosis. Surface labeling was performed for V5 (green) and HA (magenta). In a separate condition, FAM19A1-V5 was coexpressed with Nrxn1β. The mean intensity of V5 pixels colocalized with HA pixels was normalized to the mean intensity of HA. Surface FAM19A1-V5 levels were lower when recombinant FAM19A1-V5 was added to Nrxn1β-, Nrxn1β-ΔCysL-, Nrxn1β-ΔCHO-, or PDGFR TM-expressing cells as compared with when FAM19A1-V5 and Nrxn1β were coexpressed (F). Data are means ± SEM ( n = 3 independent experiments). Statistical analyses were performed using a repeated-measures one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*, P < 0.05; **, P < 0.01). (G) Purified recombinant (recomb.) Nrxn1β-ECD-ΔCHO-Myc-6xHis ( ; magenta) was mixed with separately purified recombinant FAM19A1-V5-6xHis (A–D; green) for 4 h at 4°C and analyzed by non-reducing and reducing SDS-PAGE. Comigration was not observed under either condition. **, FAM19A1 homodimers; *, FAM19A1 monomers. (H–J) Purified recombinant FAM19A1 containing the endogenous signal peptide, as well as V5 and 6xHis tags, mostly exists as a monomer (*), with disulfide-mediated dimers detected in the input fraction in non-reducing (I), but not reducing (J), Coomassie-blue SDS-gels. Peak 2 consists of FAM19A1 monomers (H), while peak 1 likely contains an unspecific ∼150 kD protein. Monomeric purified recombinant FAM19A1 from fractions delineated by dotted lines (I and J) was used for absolute mass determination by ESI-MS . FAM19A1-V5-6xHis was expressed in FreeStyle 293-F cells using the BacMam system. Recombinant FAM19A1 was purified by immobilized metal affinity chromatography (IMAC) followed by SEC on a Superdex 75 column. (K–M) Purified recombinant FAM19A1 in which the endogenous signal peptide was replaced with that of Igκ, and containing a Twin-Strep tag, exists as a monomer (*), with disulfide-mediated dimers (**) detected in the input fraction and within the left shoulder of Peak 2 in non-reducing (L), but not reducing (M), Coomassie-blue SDS-gels. Putatively, concentration-dependent disulfide-mediated multimers are detected within Peak 1 (L and M). Trial purification of recombinant FAM19A1-Twin-Strep shown in . Recombinant FAM19A1-Twin-Strep was expressed in HEK293S GnTI − cells using the BacMam system and purified by batch elution from a StrepTactin column followed by SEC on a Superdex 75 column. (N and O) Side-by-side comparison of recombinant FAM19A1-Twin-Strep fractions analyzed by Coomassie-blue staining (N; same as in ) and immunoblotting for Twin-Strep under reducing SDS-PAGE (O) confirms the predominance of FAM19A1 monomers within the input and the purity of FAM19A1 monomers within peak 2 . It also supports the existence of disulfide-mediated FAM19A1 multimers within peak 1 . (P) Predicted isoelectric points of the Nrxn1β-FAM19A1 complex and individual recombinant proteins shown in , , and (Prot pi Protein Tool), which were used to inform the purification of the complex. Note that, for all proteins, it was assumed that all cysteines form disulfide bonds. For Nrxn1β, one Man 5 GlcNAc 2 addition was assumed.

Article Snippet: The following primary antibodies were used for immunoblotting: chicken anti-c-myc (1:500; Aves Labs, ET-MY100, RRID: AB_2313514), mouse anti-V5 (1:1,000; Invitrogen, R960-25, RRID: AB_2556564), rabbit anti-V5 (1:1,000; Millipore, AB3792), rabbit anti-Strep-tag II (1:1,000; Abcam, ab76949), rabbit anti-myc (clone 71D10; 1:1,000; Cell Signaling, rabbit mAb 2278), mouse anti-HA.11 (clone 16B12; 1:1,000; BioLegend, MMS-101R, RRID: AB_256533), mouse anti-GAPDH (clone 6C5; 1 h incubation at RT; 1:5,000; Millipore, MAB374), rabbit anti-pan-Nrxn (1–2 overnight incubation at 4°C; 1:1,000; Millipore, ABN161-I), mouse anti-GABA A R β2/3 (clone 62-3G1; 1:500; NeuroMab, 75–363, RRID: AB_2315837), mouse anti-Δ-HS (3G10 epitope, clone F69-3G10; 1:1,000; Amsbio, 370260-S), and rabbit anti-synapsin (1:1,000; homemade, YZ6079).

Techniques: Purification, Recombinant, Transfection, Size-exclusion Chromatography, SDS Page, Binding Assay, Construct, Glycoproteomics, Modification, Concentration Assay, Labeling, Expressing, Affinity Chromatography, Strep-tag, Comparison, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

doi: 10.1083/jcb.202004164

Figure Lengend Snippet: FAM19A1 binding to Nrxn1β requires the cysteine-loop domain of Nrxn1β. (A) Schematic of Nrxn1β constructs used for deletion mapping experiments (all on the Nrxn1β SS4-SS5- -ECD backbone; Igκ SP, Igκ signal peptide; β-spec., β-Nrxn-specific sequence; LNS, sixth LNS domain [the only LNS-domain of β-neurexins]; stalk, O -glycosylated sequence separating the LNS-domain from the transmembrane region that is interrupted by the cysteine-loop (CysL) domain; N -glyc. and O -glyc., N - and O -linked glycosylation sites; HS, heparan sulfate modification site). (B) Coimmunoprecipitation (coIP) assays demonstrate that the CysL-domain of Nrxn1β is required for FAM19A1 binding. FAM19A1-V5 was coexpressed with wild-type (WT) or mutant Nrxn1β-ECDs (as Nrxn1β SS4-SS5- -ECD-Myc-6xHis constructs) in HEK293T cells. FAM19A1 was immunoprecipitated from the medium using V5 antibodies and immunoblotted as indicated. (C) Amino acid sequences of the WT Nrxn1β-ECD (top) and of the various CysL-domain mutants analyzed for FAM19A1 binding (bottom). (D) coIPs performed as described for B demonstrate that, apart from the two cysteine residues, the CysL sequence of Nrxn1β is irrelevant for FAM19A1 binding, and that insertion of a cysteine-loop sequence from GABA A R-β3 into the CysL of Nrxn1β does not impair FAM19A1 binding. Splice site A1-containing Nlgn1 was used as a negative control. (E) Immunoblotting analyses confirm that mutation of the Nrxn1β CysL-domain cysteines, but not of the actual loop itself, impairs FAM19A1 binding. The media of HEK293T cells coexpressing FAM19A1-V5 with WT or mutant Nrxn1β-ECDs were analyzed by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting (magenta, Myc; green, V5; white, overlap). Disulfide-bonded FAM19A1 dimers are detected under non-reducing conditions when FAM19A1 is coexpressed with a non-binding Nrxn mutant (CysL→A). **, FAM19A1 homodimers; *, FAM19A1 monomers. See also . (F) coIP assays of HEK293T lysates coexpressing FAM19A1-V5 with the HA-tagged GABA A R-β3, Nrxn1β, or mutant Nrxn1β lacking the CysL-domain show that GABA A -β3 receptors do not bind to FAM19A1. (G) Quantifications of FAM19A1 protein levels in input fractions of experiments shown in F demonstrate that Nrxn1β is required for high-level expression of FAM19A1. FAM19A1 protein levels were normalized to GAPDH and divided by coexpressed recombinant protein levels (i.e., GABA A R-β3, Nrxn1β, or Nrxn1β-ΔCysL normalized to GAPDH), followed by normalization to the negative control group (Nrxn1β-ΔCysL). Data are means ± SEM, n = 3 experimental replicates. Statistical analyses were performed using a one-way ANOVA with Tukey’s post hoc test for multiple comparisons (**, P < 0.01; ***, P < 0.001). Images depict representative blots from experiments that were independently replicated at least three times (B and D–G). IB, immunoblot; IP, immunoprecipitation.

Article Snippet: The following primary antibodies were used for immunoblotting: chicken anti-c-myc (1:500; Aves Labs, ET-MY100, RRID: AB_2313514), mouse anti-V5 (1:1,000; Invitrogen, R960-25, RRID: AB_2556564), rabbit anti-V5 (1:1,000; Millipore, AB3792), rabbit anti-Strep-tag II (1:1,000; Abcam, ab76949), rabbit anti-myc (clone 71D10; 1:1,000; Cell Signaling, rabbit mAb 2278), mouse anti-HA.11 (clone 16B12; 1:1,000; BioLegend, MMS-101R, RRID: AB_256533), mouse anti-GAPDH (clone 6C5; 1 h incubation at RT; 1:5,000; Millipore, MAB374), rabbit anti-pan-Nrxn (1–2 overnight incubation at 4°C; 1:1,000; Millipore, ABN161-I), mouse anti-GABA A R β2/3 (clone 62-3G1; 1:500; NeuroMab, 75–363, RRID: AB_2315837), mouse anti-Δ-HS (3G10 epitope, clone F69-3G10; 1:1,000; Amsbio, 370260-S), and rabbit anti-synapsin (1:1,000; homemade, YZ6079).

Techniques: Binding Assay, Construct, Sequencing, Glycoproteomics, Modification, Mutagenesis, Immunoprecipitation, Negative Control, Western Blot, SDS Page, Expressing, Recombinant

Journal: The Journal of Cell Biology

Article Title: Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

doi: 10.1083/jcb.202004164

Figure Lengend Snippet: The disulfide-bonded Nrxn1β-ECD/FAM19A1 complex is assembled in the secretory pathway. (A) The purified recombinant Nrxn1β-ECD/FAM19A1 complex is dissociated by disulfide-bond reduction, but not by SDS-sample buffer alone (SDS-PAGE followed by Coomassie-blue staining or immunoblotting; magenta, Myc; green, V5; white, overlap; DTT, dithiothreitol). (B) The purified recombinant Nrxn1β/FAM19A1 complex is dissociated by disulfide-bond reduction also under native conditions (native PAGE followed by Coomassie-blue staining or immunoblotting; magenta, Myc; green, V5; white, overlap; TCEP, tris(2-carboxyethyl)phosphine). (C and D) The disulfide-bonded Nrxn1β/FAM19A1 complex is formed during intracellular transport of Nrxn1β and FAM19A1 in the secretory pathway. HEK293T cells coexpressing the indicated proteins and their media were separately collected in SDS-sample buffer and immunoblotted as indicated (magenta, Myc; green, V5; white, overlap). In the absence of Nrxn1β-ECD, FAM19A1 is largely retained as a disulfide-bonded aggregate in the cells. Cells expressing both the Nrxn1β-ECD and FAM19A1 in C contain Nrxn1β-ECD in a heterodimeric complex with FAM19A1 (white band) and as a monomer with a lower molecular weight (magenta band).

Article Snippet: The following primary antibodies were used for immunoblotting: chicken anti-c-myc (1:500; Aves Labs, ET-MY100, RRID: AB_2313514), mouse anti-V5 (1:1,000; Invitrogen, R960-25, RRID: AB_2556564), rabbit anti-V5 (1:1,000; Millipore, AB3792), rabbit anti-Strep-tag II (1:1,000; Abcam, ab76949), rabbit anti-myc (clone 71D10; 1:1,000; Cell Signaling, rabbit mAb 2278), mouse anti-HA.11 (clone 16B12; 1:1,000; BioLegend, MMS-101R, RRID: AB_256533), mouse anti-GAPDH (clone 6C5; 1 h incubation at RT; 1:5,000; Millipore, MAB374), rabbit anti-pan-Nrxn (1–2 overnight incubation at 4°C; 1:1,000; Millipore, ABN161-I), mouse anti-GABA A R β2/3 (clone 62-3G1; 1:500; NeuroMab, 75–363, RRID: AB_2315837), mouse anti-Δ-HS (3G10 epitope, clone F69-3G10; 1:1,000; Amsbio, 370260-S), and rabbit anti-synapsin (1:1,000; homemade, YZ6079).

Techniques: Purification, Recombinant, SDS Page, Staining, Western Blot, Clear Native PAGE, Expressing, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

doi: 10.1083/jcb.202004164

Figure Lengend Snippet: FAM19A1 binding to Nrxn1β is dependent on the cysteine residues in the neurexin cysteine-loop domain, but the cysteine-loop domain is insufficient for FAM19A1 binding since FAM19A1 does not bind to Nrxn1γ that contains the cysteine-loop domain, whereas CA10 does bind to Nrxn1γ. (A) Extended views of the immunoblots shown in . Under non-reducing conditions, FAM19A1 (green) comigrates (white) with all Nrxn mutants (magenta) shown here except when the Nrxn cysteine-loop (CysL) is deleted (ΔCysL) or the CysL cysteines are mutated (CysL C→A). The complexes are dissociated under reducing conditions. Further, disulfide-mediated FAM19A1 dimers are evident under non-reducing conditions when FAM19A1 is coexpressed with non-binding Nrxn mutants. Finally, FAM19A1 does not comigrate with Nlgn1, and FAM19A1 levels are relatively low when coexpressed with Nlgn1 (but detectable upon immunoprecipitation; see ). (B) Coimmunoprecipitation (coIP) assays of HEK293T lysates demonstrate that HA-tagged Nrxn1γ does not bind to coexpressed V5-tagged FAM19A1, whereas it does bind to coexpressed CA10 . Consistent with the dual requirement of the DILV sequence and the CysL domain in Nrxn for CA10 binding, Nrxn1γ lacking an intact CysL and CA10 coprecipitate more weakly than intact Nrxn1γ and CA10, suggesting that the DILV sequence is sufficient for some degree of binding. V5-tagged FAM19A1 or CA10 were coexpressed with the full-length wild-type (WT) or mutant Nrxn1γ, Nrxn1β (positive control), or the PDGFR transmembrane domain (TM; negative control) in HEK293T cells. Immunoprecipitation of HEK293T lysate was performed using HA antibodies and the immunoprecipitates were subjected to immunoblotting with antibodies to HA, V5, and GAPDH. Images depict representative blots from experiments that were independently replicated two times. (C) Same as B, except that the samples were immunoprecipitated with antibodies to V5. IB, immunoblot; IP, immunoprecipitation.

Article Snippet: The following primary antibodies were used for immunoblotting: chicken anti-c-myc (1:500; Aves Labs, ET-MY100, RRID: AB_2313514), mouse anti-V5 (1:1,000; Invitrogen, R960-25, RRID: AB_2556564), rabbit anti-V5 (1:1,000; Millipore, AB3792), rabbit anti-Strep-tag II (1:1,000; Abcam, ab76949), rabbit anti-myc (clone 71D10; 1:1,000; Cell Signaling, rabbit mAb 2278), mouse anti-HA.11 (clone 16B12; 1:1,000; BioLegend, MMS-101R, RRID: AB_256533), mouse anti-GAPDH (clone 6C5; 1 h incubation at RT; 1:5,000; Millipore, MAB374), rabbit anti-pan-Nrxn (1–2 overnight incubation at 4°C; 1:1,000; Millipore, ABN161-I), mouse anti-GABA A R β2/3 (clone 62-3G1; 1:500; NeuroMab, 75–363, RRID: AB_2315837), mouse anti-Δ-HS (3G10 epitope, clone F69-3G10; 1:1,000; Amsbio, 370260-S), and rabbit anti-synapsin (1:1,000; homemade, YZ6079).

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Sequencing, Mutagenesis, Positive Control, Negative Control

Journal: The Journal of Cell Biology

Article Title: Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

doi: 10.1083/jcb.202004164

Figure Lengend Snippet: FAM19A1 expression depends on neurexins and alters the levels and apparent size of α-neurexins. (A) Representative immunoblots illustrating that Nrxns are required for stable FAM19A1 expression and that FAM19A1 expression changes the apparent size and levels of α-neurexins. Cell-surface proteins of hippocampal cultures (prepared as described for ) were biotinylated at DIV16 and purified using streptavidin. Input, flowthrough, and biotinylated fractions were analyzed by immunoblotting. Two exposures of the neurexin immunoblot are shown to illustrate various molecular weight species. Red asterisks, non-specific bands detected using the pan-neurexin antibody ABN161-I. (B) Deletion of all neurexins decreases total and surface, but not intracellular, FAM19A1 levels, demonstrating that neurexins enable surface-transport of FAM19A1. (C) FAM19A1 reduces total and intracellular, but not surface, α-neurexin levels. (D) FAM19A1 decreases the relative abundance of highly glycosylated “high molecular weight” α-neurexins and increases the relative abundance of less glycosylated “low molecular weight” α-neurexins in all fractions. (E) FAM19A1 has no effect on heparan sulfate (HS)-modified (HS + ) β-neurexin levels, identified as high molecular weight β-neurexin variants that disappear with the addition of heparinases (see ). (F) FAM19A1 has no effect on total or intracellular, but increases surface, HS – β-neurexin levels. Data in B–F are means ± SEM ( n = 3 independent cultures with at least three biological samples pooled per condition). Data in B, C, E, and F are normalized to GAPDH and the control group within each experimental replicate. Data in D are normalized to total α-neurexin levels within each group. Statistical analyses were performed using two-tailed one-sample t tests (B, C, E, and F) or paired t tests (D; *, P < 0.05; **, P < 0.01).

Article Snippet: The following primary antibodies were used for immunoblotting: chicken anti-c-myc (1:500; Aves Labs, ET-MY100, RRID: AB_2313514), mouse anti-V5 (1:1,000; Invitrogen, R960-25, RRID: AB_2556564), rabbit anti-V5 (1:1,000; Millipore, AB3792), rabbit anti-Strep-tag II (1:1,000; Abcam, ab76949), rabbit anti-myc (clone 71D10; 1:1,000; Cell Signaling, rabbit mAb 2278), mouse anti-HA.11 (clone 16B12; 1:1,000; BioLegend, MMS-101R, RRID: AB_256533), mouse anti-GAPDH (clone 6C5; 1 h incubation at RT; 1:5,000; Millipore, MAB374), rabbit anti-pan-Nrxn (1–2 overnight incubation at 4°C; 1:1,000; Millipore, ABN161-I), mouse anti-GABA A R β2/3 (clone 62-3G1; 1:500; NeuroMab, 75–363, RRID: AB_2315837), mouse anti-Δ-HS (3G10 epitope, clone F69-3G10; 1:1,000; Amsbio, 370260-S), and rabbit anti-synapsin (1:1,000; homemade, YZ6079).

Techniques: Expressing, Western Blot, Purification, Molecular Weight, High Molecular Weight, Modification, Control, Two Tailed Test

Journal: Cell Cycle

Article Title: Budding yeast CENP-A Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

doi: 10.1080/15384101.2017.1380129

Figure Lengend Snippet: Genome-wide two-hybrid screen for CENP-ACse4 identified Sgo1 as an interacting partner. (A) Schematic of yeast two-hybrid assay to identify interactors of CENP-ACse4 used here as a bait. BD = binding domain, AD = activating domain, UAS = upstream activating sequence. GAL4 gene, a transcription factor, produces BD and AD protein products, which are required for transcription of the reporter gene. GAL4-BD+CENP-ACse4 and GAL4-AD+Prey (yeast genes) fusion constructs were used. These fusion proteins alone cannot activate reporter gene transcription; however, expression of both fusion proteins allows interaction between CENP-ACse4 and prey protein leading to transcription of the reporter gene. (B) List of genes showing statistically significant two-hybrid interaction with CENP-ACse4.

Article Snippet: Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc ; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-A Cse4 ; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies.

Techniques: Genome Wide, Two Hybrid Screening, Y2H Assay, Binding Assay, Sequencing, Construct, Expressing

Journal: Cell Cycle

Article Title: Budding yeast CENP-A Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

doi: 10.1080/15384101.2017.1380129

Figure Lengend Snippet: CENP-ACse4 interacts with Sgo1 and sgo1-NT in vivo and in vitro. (A) Sgo1 prey constructs was tested against the CENP-ACse4 bait construct. Sgo1 prey resulted in increased growth compared with vector (pADC) or CENP-ACse4 prey when tested against the CENP-ACse4 FL bait construct. (B) CENP-ACse4 interacts in vivo with Sgo1 and sgo1-NT. Wild type strain (SGO1-MYC, YMB10165), sgo1-NT-MYC (YMB10160) expressing CENP-ACse4 from galactose inducible promoter (GAL1) were grown in YEP with 2% galactose + 2% raffinose at 25°C. Untagged strain (OCF1533-4B), SGO1-MYC (AMY905), and sgo1-NT-MYC (YMB10076) were used as a control. Cell extracts were prepared for immunoprecipitation experiments using anti-HA agarose antibodies (A2095, Sigma-Aldrich). Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-ACse4; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies. IN = input, and IP = immunoprecipitated samples. (C) A minimal prey construct of AD-sgo1-NT132 (amino acids 1–132) resulted in two-hybrid positive interactions with CENP-ACse4-DBD, sgo1-NT (amino acids 1–150) and Rts1-DBD. Scm3-AD was also positive with CENP-ACse4-DBD. Two-hybrid selection plates were SD–HLT and supplemented with 3-aminotriazole (1 or 3 mM). (D) Affinity pull-down of recombinant proteins shows that GST-sgo1-NT132 immobilized on glutathione beads can interact and pull-down His6-CENP-ACse4 octasome from E. coli lysate. IN = input (1% of the input run on a separate gel). *Represents degraded or proteolyzed products. Pull-down experiments were performed three times with similar results.

Article Snippet: Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc ; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-A Cse4 ; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies.

Techniques: In Vivo, In Vitro, Construct, Plasmid Preparation, Expressing, Control, Immunoprecipitation, Western Blot, Selection, Recombinant

Journal: Cell Cycle

Article Title: Budding yeast CENP-A Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

doi: 10.1080/15384101.2017.1380129

Figure Lengend Snippet: Sgo1 and sgo1-NT associate with core CEN in a cell cycle dependent manner. Wild type (SGO1-MYC, AMY905), sgo1-NT-MYC (YMB10076), and untagged control (OCF1533-4B) were grown in YPD to logarithmic phase (LOG) at 25°C, and synchronized in G1 with alpha factor (3 µM), S-phase with HU (0.2 M), and in G2/M with nocodazole (20 µg/mL) at 25°C for 2 hours. ChIP was performed using anti-Myc agarose beads (A7470, Sigma-Aldrich). Enrichment at CEN and at a negative control region ACT1 was determined by qPCR and is shown as % input. Average from three biological replicates ± standard error is shown. **p value <0.01, Student's t test. (A) Western blotting showing expression of Sgo1-Myc and sgo1-NT-Myc in LOG or various stages of the cell cycle. Antibodies used were: anti-Myc (Sgo1 or sgo1-NT; a-14, sc-789, Santa Cruz Biotechnology), and anti-Tub2 (loading control) antibodies. (B) FACS profiles show DNA content representing various stages of the cell cycle. (C) ChIP-qPCR showing enrichment levels of Sgo1-Myc and sgo1-NT-Myc at CEN1 in LOG phase or at various stages of the cell cycle. (D) ChIP-qPCR showing enrichment levels of Sgo1-Myc and sgo1-NT-Myc at CEN3 in LOG phase or at various stages of the cell cycle. (E) ChIP-qPCR showing enrichment levels of Sgo1-Myc and sgo1-NT-Myc at CEN5 in LOG phase or at various stages of the cell cycle. (F) ChIP-qPCR showing enrichment levels of Sgo1-Myc and sgo1-NT-Myc at a negative control region ACT1 in LOG phase or at various stages of the cell cycle.

Article Snippet: Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc ; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-A Cse4 ; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies.

Techniques: Control, Negative Control, Western Blot, Expressing, ChIP-qPCR

Journal: Cell Cycle

Article Title: Budding yeast CENP-A Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

doi: 10.1080/15384101.2017.1380129

Figure Lengend Snippet: sgo1-NT associates with core CEN but not with peri-CEN chromatin. (A) Schematic of CEN and peri-CEN regions on chromosome III. Centromere (CEN3, violet oblong), cohesin associated regions (CARs; green triangle with vertical lines) are shown. CEN is located at 114 kb; peri-CEN regions 115 (1 kb from CEN), 112 (2 kb from CEN), 134 (20 kb from CEN), and chromosome arm CAR 261 (147 kb from CEN) were examined. (B) Association of Sgo1-Myc and sgo1-NT-Myc at CEN and peri-CEN regions. ChIP samples from cells synchronized in G2/M as described in Figure 3 were used. Enrichment at CEN3 and peri-CEN regions was determined by qPCR and is shown as % input. Average from three biological replicates ± standard error is shown.

Article Snippet: Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc ; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-A Cse4 ; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies.

Techniques:

Journal: Cell Cycle

Article Title: Budding yeast CENP-A Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

doi: 10.1080/15384101.2017.1380129

Figure Lengend Snippet: Association of Sgo1 and sgo1-NT with core CEN requires CENP-ACse4. Wild type strain (SGO1-MYC, YMB10165), and sgo1-NT-MYC (YMB10160) expressing HA-tagged CENP-ACse4 from galactose inducible promoter (GAL1) were grown in YEP with 2% galactose + 2% raffinose at 25°C (Cse4-ON). Untagged strain (OCF1544-4B) was used as a control. Cells were collected, washed in dH2O and grown at 25°C in YEP with 2% glucose for 3 hours to Shut-off the expression of CENP-ACse4 (Cse4-OFF). Samples were collected for DNA content, protein extraction, and ChIP analyses. (A) CENP-ACse4 protein levels are not detectable upon its depletion. Western blot analysis was carried out on whole cell protein extracts prepared from cultures grown under Cse4-ON and Cse4-OFF conditions as described above. Blots were probed with anti-HA (CENP-ACse4; clone 12CA5, Roche Molecular Systems), and anti-Myc (Sgo1-Myc or sgo1-NT-Myc; a-14, sc-789, Santa Cruz Biotechnology) antibodies. (B) Depletion of CENP-ACse4 causes synchronization of cells in G2/M phase of the cell cycle as revealed by FACS analysis. (C) CEN levels of CENP-ACse4 are reduced upon its depletion. ChIP for CENP-ACse4 was carried out using anti-HA agarose beads (A2095, Sigma-Aldrich). Enrichment at CEN1, CEN3, CEN5, and ACT1 (negative control) was determined by qPCR and is shown as % input. Average from three biological replicates ± standard error is shown. **p value <0.01, *p value <0.05, Student's t test. (D) Sgo1 and sgo1-NT fail to remain associated with CEN chromatin upon depletion of CENP-ACse4. ChIP for Sgo1-Myc and sgo1-NT-Myc was performed using anti-Myc agarose beads (A7470, Sigma-Aldrich). Enrichment at CEN1, CEN3, CEN5, and ACT1 (negative control) was determined by qPCR and is shown as % input. Average from three biological replicates ± standard error is shown. **p value <0.01, *p value <0.05, Student's t test.

Article Snippet: Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc ; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-A Cse4 ; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies.

Techniques: Expressing, Control, Protein Extraction, Western Blot, Negative Control

Journal: Cell Cycle

Article Title: Budding yeast CENP-A Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

doi: 10.1080/15384101.2017.1380129

Figure Lengend Snippet: The N-terminus of Sgo1 (sgo1-NT) is required for faithful chromosome segregation. (A) Schematic of full-length SGO1 and its mutant alleles. sgo1-NT includes amino acids residues 1–150 (deletion of amino acids 151–591), whereas, sgo1-CT includes amino acids residues 151–591 (deletion of amino acids 2–150). Symbols: CC denotes the N-terminus coiled coil domain (amino acids 43–87); B denotes the C-terminus basic SGO1 motif (amino acids 366–390), and pink vertical line represents destruction box (amino acids 494–498). (B) sgo1-NT is required for faithful chromosome segregation independently of C-terminus basic domain. Frequency of CF loss in wild type (SGO1; YPH1018), sgo1Δ (YMB10224), sgo1-CT (YMB10221), and sgo1-NT (YMB10225) strains was determined as described in Materials and Methods. At least 1000 colonies from three independent transformants were counted. Average from three biological experiments ± standard error. Values sharing the same letter are not significantly different at a 5% level based on the analysis of variance (p > 0.05).

Article Snippet: Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc ; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-A Cse4 ; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies.

Techniques: Mutagenesis

Journal: Cell Cycle

Article Title: Budding yeast CENP-A Cse4 interacts with the N-terminus of Sgo1 and regulates its association with centromeric chromatin

doi: 10.1080/15384101.2017.1380129

Figure Lengend Snippet: List of yeast strains, plasmids, and primers used in this study.

Article Snippet: Eluted proteins were analyzed by Western blotting with anti-Myc (Sgo1-Myc or sgo1-NT-Myc ; a-14, sc-789, Santa Cruz Biotechnology), anti-HA (CENP-A Cse4 ; H6908, Sigma Aldrich), and anti-Tub2 (loading control) antibodies.

Techniques: Plasmid Preparation, Clone Assay, Binding Assay, Cloning

Journal: Cancers

Article Title: A DNA Methylation-Dependent NOP56/MYC Positive Feedback Loop Promotes the Proliferation and Migration of Non-Small Cell Lung Cancer Through Regulating Ribosome Biogenesis

doi: 10.3390/cancers18050751

Figure Lengend Snippet: MYC mediates oncogenic role on NOP56 in NSCLC. ( A ) GSEA plot of MYC targets in NOP56-depleted A549 cells compared with control A549 cells. ( B ) qPCR analysis of MYC target gene expression in NOP56-depleted NSCLC cells or control. ( C ) Western blot analysis of MYC expression in NOP56-depleted NSCLC cells or control. ( D ) qPCR analysis of MYC expression in NOP56-depleted NSCLC cells or control. ( E ) Western blot analysis of MYC expression in MYC-depleted NSCLC cells or control. ( F – I ) CCK-8 assays, colony formation assays, transwell assays and wound healing assays in NSCLC cells with NOP56 overexpression or MYC knockdown. The data are shown as mean ± SD. Scale bar, 100 µM, * means ovNCS + siNC vs. ovNOP56 + siNC, * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t test, # means ovNOP56 + siNC vs. ovNOP56 + siMYC, # p < 0.05, ## p < 0.01, by Student’s t test.

Article Snippet: Membranes were blocked with 5% nonfat milk and probed with primary antibodies at 4 °C overnight with primary antibodies against MYC (49 kDa, 1:2000, 10828-1-AP, Proteintech, Rosemont, IL, USA), NOP56 (66 kDa, 1:1000, A302-720A-T, Thermo, Waltham, MA, USA), or GAPDH (36 kDa, 1:5000, 60004-1-Ig, Proteintech, Rosemont, IL, USA).

Techniques: Control, Targeted Gene Expression, Western Blot, Expressing, CCK-8 Assay, Over Expression, Knockdown

Journal: Cancers

Article Title: A DNA Methylation-Dependent NOP56/MYC Positive Feedback Loop Promotes the Proliferation and Migration of Non-Small Cell Lung Cancer Through Regulating Ribosome Biogenesis

doi: 10.3390/cancers18050751

Figure Lengend Snippet: NOP56 regulates MYC through IRES-dependent translation. ( A ) RTL-P assays were conducted to detect the 2′-O methylation level of rRNA in NSCLC cells with NOP56 overexpression or control. ( B ) OP-Puro incorporation assay was conducted to detect the protein synthesis rate. ( C ) The bi-cistronic luciferase reporter was constructed as above and the Poliovirus (PV) IRES activity was calculated as the ratio of firefly luciferase activity over renilla luciferase activity. ( D ) The bi-cistronic luciferase reporter was constructed as above and the IRES-dependent translation (Fluc/Rluc) from IRES elements of MYC was measured. The data are shown as mean ± SD. Scale bar, 100 µM, * p < 0.05, ** p < 0.01 by Student’s t test.

Article Snippet: Membranes were blocked with 5% nonfat milk and probed with primary antibodies at 4 °C overnight with primary antibodies against MYC (49 kDa, 1:2000, 10828-1-AP, Proteintech, Rosemont, IL, USA), NOP56 (66 kDa, 1:1000, A302-720A-T, Thermo, Waltham, MA, USA), or GAPDH (36 kDa, 1:5000, 60004-1-Ig, Proteintech, Rosemont, IL, USA).

Techniques: Methylation, Over Expression, Control, Luciferase, Construct, Activity Assay

Journal: Cancers

Article Title: A DNA Methylation-Dependent NOP56/MYC Positive Feedback Loop Promotes the Proliferation and Migration of Non-Small Cell Lung Cancer Through Regulating Ribosome Biogenesis

doi: 10.3390/cancers18050751

Figure Lengend Snippet: MYC contributes to NOP56 transcriptional regulation. ( A – C ) Correlation between NOP56 mRNA and MYC mRNA expression in TCGA-LUSC, TCGA-LUAD and GSE30219 datasets. ( D ) The predicted MYC binding sites in the NOP56 promoter region. ( E ) qPCR analysis of NOP56 expression in MYC-depleted NSCLC cells or control. ( F ) The luciferase reporter was constructed as above and the transcription (Fluc) from WT/MUT promoter elements of MYC was measured. ( G ) ChIP assay was conducted to confirm the MYC binding sites in NOP56 promoter. The data are shown as mean ± SD. ** p < 0.01, *** p < 0.001 by Student’s t test.

Article Snippet: Membranes were blocked with 5% nonfat milk and probed with primary antibodies at 4 °C overnight with primary antibodies against MYC (49 kDa, 1:2000, 10828-1-AP, Proteintech, Rosemont, IL, USA), NOP56 (66 kDa, 1:1000, A302-720A-T, Thermo, Waltham, MA, USA), or GAPDH (36 kDa, 1:5000, 60004-1-Ig, Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Binding Assay, Control, Luciferase, Construct