Journal: eLife

Article Title: Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B

doi: 10.7554/elife.46840

Figure Lengend Snippet: Figure 3. The heparan sulfate biosynthesis pathway is required for G6b-B binding to HEK293 cells. (A) Recombinant G6b-B, produced as a monomeric biotinylated protein and conjugated to streptavidin-PE to generate an avid probe, binds to HEL, HEK293 and COLO-320-HSR cells. (B) A genome-wide loss-of-function approach identifies the HS biosynthesis pathway as the factor required to mediate the binding of recombinant G6b-B to HEK293 cells (left panel). X- and y-axis represent the log-fold-change (LFC) and robust rank aggregation (RRA) score calculated using the MAGeCK software, respectively. Circles represent individual genes and sizes represent the false-discovery rate (FDR): large circle = FDR < 1%, small circle = 1% < FDR < 5%. Genes with FDR < 5% are color coded according to their functional annotation and genes corresponding to the HS biosynthesis pathway are additionally named. The HS biosynthesis pathway is depicted in the right panel with the genes identified in the loss-of-function approach highlighted. Similar results were obtained in HEL cells (not shown). (C) G6b-B binding to HEK293 cells was measured by flow cytometry in the presence or absence Figure 3 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.46840 28 of 43 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti rat IgG Alexa 546 (goat polyclonal) Invitrogen #A-11081; RRID:AB_141738 (1:300); used for IF staining of BM Antibody anti-actin (mouse monoclonal) Sigma-Aldrich #A4700, clone AC-40; RRID:AB_476730 (1:1000) Antibody Anti-a-tubulin (mouse monoclonal) Sigma-Aldrich #T6199, clone DM1A; RRID:AB_477583 (1:1000) Antibody anti-GAPDH (rabbit monoclonal) Cell Signaling Technology #2118, clone: 14C10; RRID:AB_561053 (1:10) dilution, on 0.05 mg/ml lysates for Wes Antibody anti-Src p-Tyr418 (rabbit polyclonal) Sigma-Aldrich #44660G; RRID:AB_1500523 (1:10) dilution, on 0.05 mg/ml lysates for Wes Antibody anti-Shp1 p-Tyr564 (rabbit monoclonal) Cell Signaling Technology #8849, clone: D11G5; RRID:AB_11141050 (1:10) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-Shp2 p-Tyr542 (rabbit polyclonal) Cell Signaling Technology #3751; RRID:AB_330825 (1:10) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-Shp2 p-Tyr580 (rabbit polyclonal) Cell Signaling Technology #3703; RRID:AB_2174962 (1:10) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-Syk p-Tyr525/6 (rabbit polyclonal) Cell Signaling Technology #2711; RRID:AB_2197215 (1:50) dilution, on 0.2 mg/ml lysates for Wes Antibody anti-SH-PTP1/Shp-1 (rabbit polyclonal) Santa Cruz sc-287 (C19); RRID:AB_2173829 (1:1000) Antibody anti-SH-PTP2/Shp-2 (rabbit polyclonal) Santa Cruz sc-280 (C18); RRID:AB_632401 (1:1000) Antibody anti-phosphotyrosine (mouse monoclonal) Merck-Millipore 05–321, clone 4G10; RRID:AB_309678 (1:1000) Antibody anti-human G6b-B (mouse monoclonal) other clone 17–4 10 mg/ml, custom-made lab reagent Peptide, recombinant protein purified human IgG-Fc fragment Bethyl Laboratories P80-104 Peptide, recombinant protein recombinant Mouse Syndecan-2/CD362 protein, CF R&D Systems 6585-SD-050 Peptide, recombinant protein recombinant human Agrin protein, N-terminal, CF R&D Systems 8909-AG-050 Peptide, recombinant protein rec. human laminin 111 Biolamina LN111-02 Peptide, recombinant protein rec. human laminin 411 Biolamina LN411-02 Peptide, recombinant protein rec. human laminin 421 Biolamina LN421-02 Peptide, recombinant protein rec. human laminin 511 Biolamina LN511-02 Continued on next page Vögtle et al. eLife 2019;8:e46840.

Techniques: Binding Assay, Recombinant, Produced, Genome Wide, Software, Functional Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Dyrk1a gene dosage controls bipolar cell development and retinal connectivity

doi: 10.64898/2026.03.15.710015

Figure Lengend Snippet: (A) Schematic representation of electroretinogram (ERG) components. An initial negative deflection (a-wave) is observed upon photoreceptor stimulation (arrow) that is followed by a subsequent positive deflection (b-wave) that corresponds to bipolar cell activity. ERG waveform quantification involves measuring the amplitude (μV) and latency (ms) of the a- and b-waves to increasing stimulus intensities (cd.s/m 2 ) and thus provides an evaluation of retinal function. (B) Representative dark-adapted scotopic ERG waveforms recorded from Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in response to a single flash stimulus (log 1.48 cd.s/m 2 ). Comparable a-wave responses are observed for both groups while b-wave amplitudes are reduced in heterozygous animals. Thick lines represent genotype-mean responses while thin lines represent individual mice. (C – D) Quantifcation of photoreceptor amplitude (C) and latency (D) responses in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in repsonse to a series of increasing scotopic flash stimuli (log cd.s/m 2 ). No significant differences are observed between the groups. (E – F) Quantification of bipolar cell amplitude (E) and latency (F) responses in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animal (6 – 8 weeks) towards increasing scotopic flash stimuli (log cd.s/m 2 ). Heterozygous animals have a significant reduction in bipolar cell amplitude response at higher stimulus intensities (E) albeit with similar latency (F). (G) Representative light-adapted photopic ERG waveforms recorded from Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in response to a single UV flash stimulus (log 1.00 cd.s/m 2 ). Comparable a-wave responses are observed for both groups while b-wave amplitudes are reduced in heterozygous animals. Thick lines represent genotype-mean responses while thin lines refer to individual mice. (H) Quantification of bipolar cell amplitude response in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animal (6 – 8 weeks) in response to an increasing photopic UV stimulus intensity series (log cd.s/m 2 ). Heterozygous animals have a significant reduction in bipolar cell amplitude response at higher stimulus intensities. (I) Representative light-adapted photopic ERG waveforms recorded from Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animals (6 – 8 weeks) in response to a single green flash stimulus (log 1.48 cd.s/m 2 ). Comparable a-wave responses are observed for both groups while b-wave amplitudes are reduced in heterozygous animals. Thick lines represent genotype-mean responses while thin lines refer to individual mice. (J) Quantification of bipolar cell amplitude response in Lhx2-Cre ( n = 3) and Lhx2-Cre:Dyrk1a +/f ( n = 3) animal (6 – 8 weeks) in response to a photopic green stimulus intensity series (log cd.s/m 2 ). Heterozygous animals have a significant reduction in bipolar cell amplitude response at higher stimulus intensities. (K – R) Representative immunohistochemical staining of coronal eye sections taken from Lhx2-Cre and Lhx2-Cre:Dyrk1a +/f adult mice (6 – 8 weeks) across four designated dorsoventral quadrants (D1, D2, V3 and V4) spanning the entire retina relative to the optic nerve head. OPL ribbon synapse assemblies appear comparable in both groups (K – R, arrowheads) and were composed of dense photoreceptor terminals (vGlut1 + ) that overlapped with well organised bipolar cell dendritic processes (PKCα + ). Additionally, the linear alignment of all ribbon junctions (Pikachurin + ) indicated intact photoreceptor and bipolar cell synapses. All data represents the mean ± SEM. Statistical differences were calculated using a two-way ANOVA followed by a Sidak’s multiple comparison test. p-values are denoted as follows: **p≤ 0.01, ***p≤ 0.001 and ****p≤ 0.0001. Scale bar: (K – R) 5 µm. Abbreviations: D, dorsal; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; V, ventral.

Article Snippet: The following primary antibodies and dilutions were used in this study: Calbindin (1:300, Sigma-Aldrich, #C9848); Calretinin (1:1000, Swant, #CG1); cleaved Caspase3 (Asp175) (1:500, Cell Signalling, #9661S); chAT (1:100, Millipore, #AB144P); Chx10 (1:50, SCBT, #SC365519); Dyrk1a (1:100, Novus, #NBP1-84032); Isl1/2 (recognises both Isl1 and Isl2 proteins) (1:500; DSHB, #39.4D5); M-opsin (1:500, Proteintech, #30975-1-AP); Pax6 (1:100, DSHB, #Pax6); Pikachurin (1:200, Proteintech, #14578-1-AP); Rhodopsin (1:500, Millipore, #MABN15); S-opsin (1:500, Proteintech, #24660-1-AP); TH (1:300, Millipore, #AB152); vGlut1 (1:2000, Millipore, #AB5905).

Techniques: Activity Assay, Immunohistochemical staining, Staining, Comparison