Journal: Scientific Reports

Article Title: Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability

doi: 10.1038/s41598-022-15784-5

Figure Lengend Snippet: Hevin mutants lacking the EF-hand motif activate the UPR signaling caused by abnormal trafficking. ( A ) Immunoblotting of Hevin WT and mutants (ΔEF and N11). Neuro-2a cells were transfected for 48 h with expression vectors for Hevin WT or mutants (ΔEF or N11). The cell lysates and conditioned medium were subjected to immunoblot analysis using anti-Hevin and Tubulin antibodies. ( B ) Immunostaining of Hevin and mutants (ΔEF and N11). HeLa cells were transfected for 24 h with expression vectors for Hevin and mCherry-ER and subjected to immunocytochemistry. Scale bar: 10 μm. ( C ) Quantification of Pearson’s correlation coefficient as the degree of colocalization in the panel B. n = 20 cells, mean ± standard error of the mean (SEM), ** p < 0.01, *** p < 0.001 versus Hevin WT, one-way analysis of variance (ANOVA) Dunnett’s test. ( D , E ) Total RNAs isolated from the Hevin-overexpressed Neuro-2a cells were conducted with qPCR analysis for measurement of Bip and Chop mRNAs. 5S rRNAs were used for normalization. n = 5; mean ± SEM; * p < 0.05, ** p < 0.01 versus Hevin WT, one-way ANOVA Dunnett’s test calculated using the ΔCt value. ( F ) Immunoblotting of BIP, Hevin, and Tubulin. HEK293T cells were transfected for 72 h with expression vectors for Hevin WT or mutants (ΔEF or N11). Cells were stimulated with 500 nM Thapsigarging (Tg) for 24 h. The cells were then subjected to immunoblot analysis using anti-BIP, Hevin and Tubulin antibodies.

Article Snippet: For immunoblotting goat anti-Hevin (1:1000, R&D Systems, Cat# AF2836), mouse anti-Tubulin (1:1000, Sigma, DM1A), rabbit anti-BIP (1:1000, Cell Signaling, CB0B12), and mouse anti-GST (1:500, Santa Cruz, B14) antibodies were used as primary antibodies.

Techniques: Western Blot, Transfection, Expressing, Immunostaining, Immunocytochemistry, Isolation

Journal: Scientific Reports

Article Title: Autism-associated mutation in Hevin/Sparcl1 induces endoplasmic reticulum stress through structural instability

doi: 10.1038/s41598-022-15784-5

Figure Lengend Snippet: ASD-associated W647R mutant of Hevin activates the UPR signaling. ( A ) Location of ASD-associated Hevin mutations. ( B ) Amino acid of the EF-hand motif in each species. The human Trp 647 is highly conserved. Cyan indicates the EF-hand motif, Magenta indicates non-conserved amino acid. ( C ) Immunoblotting of Hevin WT and mW633R mutant. Neuro-2a cells were transfected with expression vectors and collected at the indicated times. The upper panel indicates the experimental schedule. The transfection efficiencies were confirmed by observing co-expressed EGFP fluorescence. The cell lysates and conditioned medium were subjected to immunoblot analysis using anti-Hevin and Tubulin antibodies. The secretion rate of the Hevin WR mutant was delayed more than that of WT. CM; conditioned medium, TCL: total cell lysate ( D ) Immunostaining of Hevin WT and mW633R mutant. HeLa cells were transfected for 24 h with expression vectors for Hevin and mCherry-ER and subjected to immunocytochemistry. Scale bar: 10 μm. ( E ) Quantification of Pearson’s correlation coefficient as the degree of colocalization in the panel D. n = 15 cells, mean ± SEM, *** p < 0.001 by Student’s t- test. ( F ) Immunostaining of Hevin and mW633R mutant. HeLa cells were transfected for 24 h with expression vectors for Hevin and subjected to immunocytochemistry. Scale bar: 10 μm. ( G ) Quantification of Manders’ coefficient as the degree of colocalization of Hevin with GM130 in the panel F. n = 20 cells, mean ± SEM, *** p < 0.001 by Student’s t- test. ( H , I ) Total RNAs isolated from the Hevin-overexpressed Neuro-2a cells were conducted with qPCR analysis for measurement of Bip and Chop mRNAs. 5S rRNA were used for normalization. n = 5; ** p < 0.01, *** p < 0.001 versus Hevin WT, one-way ANOVA Dunnett’s test calculated p -value using the ΔCt value. ( J ) Immunoblotting of BIP, Hevin, and Tubulin. Neuro-2a cells were transfected for 72 h with expression vectors for Hevin WT or mW633R mutants. The cell lysates were then subjected to immunoblot analysis using anti-BIP, Hevin, and Tubulin antibodies. ( K ) Schematic structure of GST-Hevin. ( L ) Pull down of GST-Hevin and endogenous BIP in HEK293T cells. GST-Hevin was precipitated with Glutathione Sepharose beads and immunoblotted with anti-GST, BIP, and Tubulin antibodies.

Article Snippet: For immunoblotting goat anti-Hevin (1:1000, R&D Systems, Cat# AF2836), mouse anti-Tubulin (1:1000, Sigma, DM1A), rabbit anti-BIP (1:1000, Cell Signaling, CB0B12), and mouse anti-GST (1:500, Santa Cruz, B14) antibodies were used as primary antibodies.

Techniques: Mutagenesis, Western Blot, Transfection, Expressing, Fluorescence, Immunostaining, Immunocytochemistry, Isolation

Journal: Frontiers in Molecular Neuroscience

Article Title: Altered Developmental Expression of the Astrocyte-Secreted Factors Hevin and SPARC in the Fragile X Mouse Model

doi: 10.3389/fnmol.2017.00268

Figure Lengend Snippet: Hevin expression is altered at postnatal day (P) 14 in the cortex of Fmr1 knock-out (KO) mice. (A) Cultured cortical astrocytes co-labeled with anti-glial fibrillary acidic protein (GFAP; green) and anti-hevin (red) after 2 days in vitro . Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Images were obtained using a 40x objective with a Zeiss Axioimager M2. Scale bars = 50 μm. (B) Representative western blots showing hevin (~130 kDa) in cortical samples (30 μg of protein per lane) from P7, P14 and P21 WT and Fmr1 KO mice, as well as the corresponding total protein within each lane. Negative controls that were run using P14 WT whole cortical tissue with either no primary antibody or no secondary antibody are shown. (C–E) Hevin expression in the cortex of WT (black; n = 8) and Fmr1 KO (white; n = 8) mice at P7, P14 and P21. Bands representing hevin were normalized against the total protein within the same lane on the membrane, and were then expressed as a percent of the average level of hevin in the WT group. (F) Hevin expression in cortical astrocytes isolated from P14 WT (black; n = 4) and Fmr1 KO (white; n = 4) mice. Immediately to the left of the graph is shown a representative Western blot with bands corresponding to hevin from P14 WT and Fmr1 KO cortical astrocyte samples (10 μg of protein per lane), as well as the corresponding total protein. Statistical differences were denoted with either a single asterisk, P < 0.05, or a double asterisks, P < 0.01.

Article Snippet: The following antibodies were used: rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; catalog#: Z0334; Dako, Burlington, ON, Canada), chicken anti-GFAP (1:2000; catalog#: CH22102; Neuromics, Minneapolis, MN, USA) rabbit anti-hevin antibody (1:100; catalog#: bs-6110R; Bioss, Woburn, MA, USA), goat anti-SPARC antibody (10 μg/mL; catalog#: AF942; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Knock-Out, Cell Culture, Labeling, In Vitro, Staining, Western Blot, Isolation

Journal: The Journal of Neuroscience

Article Title: SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

doi: 10.1523/JNEUROSCI.0454-20.2020

Figure Lengend Snippet: Endogenous SPARCL1 is secreted at low levels by cultured mouse glia but is abundant in mouse serum. A, Design of intron-spanning primers for semiquantitative RT-PCR. Forward (F) and reverse (R) primers against SPARCL1 primers against SPARCL1 and β-actin (control) genes were situated in exons to distinguish between gDNA and mRNA amplification (left). Expected amplicon sizes are indicated (right). B, Most cortical SPARCL1 mRNA is expressed by glia. RT-PCR was performed on total RNA isolated from glia cultured alone or from glia cocultured with cortical neurons at DIV14. Amplification of SPARCL1 and β-actin transcripts was optimized to 25 PCR cycles to ensure that quantification was performed before saturation. SPARCL1 and β-actin transcripts from both sources were amplified either individually or in multiplexed format to estimate the relative abundance of SPARCL1 cDNA by normalization against β-actin. Analysis by electrophoresis shows specific amplification of SPARCL1 and β-actin cDNA without gDNA contamination. SPARCL1 mRNA is expressed at similar levels by pure glia and by neurons cocultured with glia, indicating that most cortical SPARCL1 is expressed by glia. SPARCL1 band intensities are normalized to β-actin band intensities within multiplexed RT-PCRs. C, Endogenous SPARCL1 protein is secreted at low levels in primary cortical cultures. Cortical glia were cultured alone (G) or with cortical neurons (N + G) for DIV14. Left, Coomassie stain showing the total protein composition of conditioned media and cell lysates taken from both types of cultures and from mouse serum. Right, Immunoblot of SPARCL1 (green) from conditioned media and cell lysates. Full-length SPARCL1 is secreted and present intracellularly (green arrow). Secreted SPARCL1 is proteolyzed into an array of fragments (light green arrows). Truncated SPARCL1 is present in serum (dark green arrow) but is undetectable in primary cortical cultures. D, Comparison of recombinant and endogenous SPARCL1 protein levels. Equal volumes of HEK293T cell supernatant containing native recombinant SPARCL1 and of conditioned medium harvested from primary cortical cultures before SPARCL1 treatment (DIV13) and 24 h after SPARCL1 treatment were analyzed by SDS-PAGE and immunoblotting. Left, Coomassie stain showing the total protein composition of the HEK cell supernatant and of the conditioned medium before (untreated) and after (treated) SPARCL1 treatment. Asterisks indicate bands corresponding in size to SPARCL1. Right, Immunoblot of recombinant SPARCL1 protein secreted by HEK cells and of SPARCL1 present in the conditioned medium before and after treatment. Dark gray arrows indicate full-length SPARCL1. Light gray arrows indicate proteolyzed SPARCL1. Bar graphs indicate mean ± SEM. Three independent cultures were analyzed. Statistical significance was evaluated by a Student's t test. ***p < 0.001; nonsignificant relations (n.s.) are indicated. For complete statistical analyses, see Extended Data Figure 1-1.

Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-SPARCL1 goat (R&D Systems; 1:5000), anti-pan-Nrxn rabbit (G394, T.C.S. laboratory, 1:500), anti-Nlgn1 mouse (SySy, 1:1000), anti-Nlgn2 mouse (SySy, 1:1000), anti-Nlgn3 rabbit (639B, T.C.S. laboratory, 1:500), and anti-β-actin mouse (A1976, Sigma Millipore, 1:2000).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation, Electrophoresis, Staining, Western Blot, Comparison, Recombinant, SDS Page

Journal: The Journal of Neuroscience

Article Title: SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

doi: 10.1523/JNEUROSCI.0454-20.2020

Figure Lengend Snippet: Recombinant SPARCL1 selectively boosts excitatory but not inhibitory synapse numbers in primary cultures of cortical neurons and glia. A, Experimental strategy. Cortical neurons and glia were cultured from P0 WT, Nrxn123, or Nlgn1234 cKO mice. At DIV3, neurons were infected with lentiviruses expressing nuclear Cre-recombinase fused to EGFP and driven by the synapsin promoter (Syn-Cre-EGFP), or nonfunctional mutant Cre (Syn-ΔCre-EGFP) as a negative control. At DIV10, neurons were transfected with β-actin-eBFP for morphologic analyses. Neurons were treated with recombinant SPARCL1 at DIV13 and analyzed by electrophysiology, immunocytochemistry, and immunoblotting at DIV14-DIV16. In the experiments described in the present figure analyzing WT neurons, cultures expressing ΔCre were examined. In all subsequent figures describing experiments on conditional neurexin and neuroligin mutants, cultures expressing ΔCre and Cre were investigated. B, Representative images showing that recombinant SPARCL1 treatment increases the excitatory synapse density in WT neurons. Images represent dendritic segments of WT neurons treated at DIV13 with the supernatants of HEK cells expressing either mClover (Control) or recombinant SPARCL1. Neurons were immunostained for vGluT1 (excitatory presynaptic marker), Homer (excitatory postsynaptic marker), and MAP2 (dendritic marker) at DIV14. C, Quantifications showing that recombinant SPARCL1 increases the density (left), but not the size of excitatory synapses (right). D, Representative images showing that recombinant SPARCL1 treatment does not alter inhibitory synapse in WT neurons. Images represent dendritic segments of WT neurons treated as described in B, and immunostained for vGAT (inhibitory presynaptic marker), gephyrin (inhibitory postsynaptic marker), and MAP2 (dendritic marker) at DIV14. E, Quantifications showing that recombinant SPARCL1 does not alter the density (left) or size (right) of inhibitory synapses. F, Representative images showing that recombinant SPARCL1 treatment does not alter the survival or dendritic morphology of WT neurons. Images represent neurons treated as described in B, but additionally sparsely transfected with eBFP to visualize dendritic arborizations of neurons. Neurons were counterstained with MAP2. G–I, Quantifications showing that recombinant SPARCL1 does not alter the neuronal cell density (G), number of primary dendrites, and dendritic branches (H), or soma size of neurons (I). J, Electrophysiological measurements of the capacitance (left) and input resistance (right) of WT neurons treated with SPARCL1 as described in B show that SPARCL1 has no significant effect on these passive electrical membrane properties. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within bars. Statistical significance was evaluated by a Student's t test. **p < 0.01; nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 2-1.

Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-SPARCL1 goat (R&D Systems; 1:5000), anti-pan-Nrxn rabbit (G394, T.C.S. laboratory, 1:500), anti-Nlgn1 mouse (SySy, 1:1000), anti-Nlgn2 mouse (SySy, 1:1000), anti-Nlgn3 rabbit (639B, T.C.S. laboratory, 1:500), and anti-β-actin mouse (A1976, Sigma Millipore, 1:2000).

Techniques: Recombinant, Cell Culture, Infection, Expressing, Mutagenesis, Negative Control, Transfection, Immunocytochemistry, Western Blot, Marker, Membrane

Journal: The Journal of Neuroscience

Article Title: SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

doi: 10.1523/JNEUROSCI.0454-20.2020

Figure Lengend Snippet: Deletion of neurexins in dissociated cultures of mouse neurons and glia neither decreases excitatory or inhibitory synapse numbers nor impairs the increase in excitatory synapse numbers produced by recombinant SPARCL1. A, Deletion of all neurexins does not significantly decrease synapse numbers and does not block excitatory synaptogenesis induced by recombinant SPARCL1. Representative images show Nrxn123, control (ΔCre), and cKO (Cre) neurons treated at DIV13 with the supernatants of HEK cells expressing either mClover (Control medium) or recombinant SPARCL1. Neurons were immunostained for VGLUT1 (excitatory puncta), VGAT (inhibitory puncta), and MAP2 at DIV14. Higher-magnification images (right) were taken from the boxed areas shown in the corresponding lower-magnification images (left). B, C, Quantifications showing that recombinant SPARCL1 increases the density of excitatory synapses (top summary graphs) but not inhibitory synapses (bottom summary graphs) in control and Nrxn123 cKO neurons. SPARCL1 did not affect the size and staining intensity of excitatory and inhibitory synapses. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within bars. *p < 0.05 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant (n.s.) relations are indicated. For complete statistical analyses, see Extended Data Figure 4-1.

Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-SPARCL1 goat (R&D Systems; 1:5000), anti-pan-Nrxn rabbit (G394, T.C.S. laboratory, 1:500), anti-Nlgn1 mouse (SySy, 1:1000), anti-Nlgn2 mouse (SySy, 1:1000), anti-Nlgn3 rabbit (639B, T.C.S. laboratory, 1:500), and anti-β-actin mouse (A1976, Sigma Millipore, 1:2000).

Techniques: Produced, Recombinant, Blocking Assay, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

doi: 10.1523/JNEUROSCI.0454-20.2020

Figure Lengend Snippet: Recombinant SPARCL1 increases spontaneous excitatory but not inhibitory synaptic transmission independent of neurexins. A, SPARCL1 increases mEPSC frequency but not amplitude independent of neurexins. Nrxn123, control (ΔCre), and cKO (Cre) neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous excitatory synaptic activity was assessed at DIV14 by mEPSC recordings in the presence of TTX and PTX. Top, Representative traces. Bottom, Summary graphs of the mEPSC frequency and mEPSC amplitude. *p < 0.05 (one-way ANOVA with Tukey's post hoc comparisons). B, SPARCL1 does not affect spontaneous inhibitory synaptic activity. Nrxn123 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous inhibitory synaptic activity was assessed at DIV14 by mIPSC recordings in the presence of TTX, CNQX, and D-AP5. Top, Representative traces. Bottom, Summary graphs of the mIPSC frequency and mIPSC amplitude. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown beside the bars. ***p < 0.001 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 5-1.

Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-SPARCL1 goat (R&D Systems; 1:5000), anti-pan-Nrxn rabbit (G394, T.C.S. laboratory, 1:500), anti-Nlgn1 mouse (SySy, 1:1000), anti-Nlgn2 mouse (SySy, 1:1000), anti-Nlgn3 rabbit (639B, T.C.S. laboratory, 1:500), and anti-β-actin mouse (A1976, Sigma Millipore, 1:2000).

Techniques: Recombinant, Transmission Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

doi: 10.1523/JNEUROSCI.0454-20.2020

Figure Lengend Snippet: Deletion of neuroligins does not significantly decrease synapse numbers and does not block excitatory synaptogenesis induced by recombinant SPARCL1. A, Deletion of all neuroligins does not significantly decrease synapse numbers and does not block excitatory synaptogenesis induced by recombinant SPARCL1. Representative images show Nlgn1234, control (ΔCre), and cKO (Cre) neurons treated at DIV13 with the supernatants of HEK cells expressing either mClover (Control medium) or recombinant SPARCL1. Neurons were immunostained for VGLUT1 (excitatory puncta), VGAT (inhibitory puncta), and MAP2 at DIV14. Higher-magnification images (right) were taken from the boxed areas shown in the corresponding lower-magnification images (left). B, C, Quantifications showing that recombinant SPARCL1 increases the density of excitatory synapses (top summary graphs) but not inhibitory synapses (bottom summary graphs) in control and Nlgn1234 cKO neurons. SPARCL1 did not substantially affect the size and staining intensity of excitatory and inhibitory synapses. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within bars. *p < 0.05; **p < 0.01 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 6-1.

Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-SPARCL1 goat (R&D Systems; 1:5000), anti-pan-Nrxn rabbit (G394, T.C.S. laboratory, 1:500), anti-Nlgn1 mouse (SySy, 1:1000), anti-Nlgn2 mouse (SySy, 1:1000), anti-Nlgn3 rabbit (639B, T.C.S. laboratory, 1:500), and anti-β-actin mouse (A1976, Sigma Millipore, 1:2000).

Techniques: Blocking Assay, Recombinant, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

doi: 10.1523/JNEUROSCI.0454-20.2020

Figure Lengend Snippet: Recombinant SPARCL1 increases spontaneous excitatory but not inhibitory synaptic transmission independent of neuroligins. A, Deletion of all neuroligins significantly reduces mEPSC frequency and amplitude but does not prevent SPARCL1 from increasing mEPSC frequency. Nlgn1234, control (ΔCre), and cKO (Cre) neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous excitatory synaptic activity was assessed at DIV14 by mEPSC recordings in the presence of TTX and PTX. Top, Representative traces. Bottom, Summary graphs of the mEPSC frequency and mEPSC amplitude. B, Deletion of all neuroligins significantly reduces mIPSC frequency and amplitude, which are unaltered by SPARCL1. Nlgn1234 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous inhibitory synaptic activity was assessed at DIV14 by mIPSC recordings in the presence of TTX, CNQX, and D-AP5. Top, Representative traces. Bottom, Summary graphs of the mIPSC frequency and mIPSC amplitude. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown beside the bars. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 7-1.

Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-SPARCL1 goat (R&D Systems; 1:5000), anti-pan-Nrxn rabbit (G394, T.C.S. laboratory, 1:500), anti-Nlgn1 mouse (SySy, 1:1000), anti-Nlgn2 mouse (SySy, 1:1000), anti-Nlgn3 rabbit (639B, T.C.S. laboratory, 1:500), and anti-β-actin mouse (A1976, Sigma Millipore, 1:2000).

Techniques: Recombinant, Transmission Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

doi: 10.1523/JNEUROSCI.0454-20.2020

Figure Lengend Snippet: SPARCL1 selectively enhances excitatory evoked neurotransmission and increases NMDAR-mediated synaptic responses independent of neuroligins. A, Deletion of all neuroligins reduces the amplitudes of evoked AMPAR-EPSCs and NMDAR-EPSCs. Treatment with recombinant SPARCL1 increases these parameters and particularly enhances the NMDAR/AMPAR ratio independent of neuroligins. Nlgn1234 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Neurons were analyzed at DIV14-DIV16 by recording EPSCs evoked by extracellular stimulation. AMPAR-EPSCs and NMDAR-EPSCs were pharmacologically isolated with PTX and monitored at −70 mV and 40 mV holding potentials, respectively. Representative traces of evoked EPSCs are shown for all conditions. B, Summary graphs of evoked AMPAR-EPSC amplitude, NMDAR-EPSC amplitude, and NMDAR/AMPAR ratio. NMDAR current amplitudes were measured 50 ms after stimulation. C, Deletion of all neuroligins reduces the amplitudes of evoked GABAR-IPSCs, whereas addition of SPARCL1 does not alter these currents. Nlgn1234 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Neurons were analyzed at DIV14-DIV16 by recording IPSCs evoked by extracellular stimulation. GABAR-IPSCs were pharmacologically isolated with D-AP5 and CNQX and monitored at −70 mV. Representative traces of evoked IPSCs are shown for all conditions. D, Summary graph of evoked GABAR-IPSC amplitude. Bar and line graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within the bars. *p < 0.05; ***p < 0.001; one-way ANOVAs and Tukey's post hoc comparisons. Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 8-1.

Article Snippet: Membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-SPARCL1 goat (R&D Systems; 1:5000), anti-pan-Nrxn rabbit (G394, T.C.S. laboratory, 1:500), anti-Nlgn1 mouse (SySy, 1:1000), anti-Nlgn2 mouse (SySy, 1:1000), anti-Nlgn3 rabbit (639B, T.C.S. laboratory, 1:500), and anti-β-actin mouse (A1976, Sigma Millipore, 1:2000).

Techniques: Recombinant, Isolation

Journal: European Journal of Human Genetics

Article Title: Autosomal dominant stromal corneal dystrophy associated with a SPARCL1 missense variant

doi: 10.1038/s41431-024-01687-8

Figure Lengend Snippet: A Pedigree structure and segregation of a SPARCL1 variant. *DNA available. WGS whole genome sequenced. SPARCL1 , NM_004684: c.334G > A; p.(Glu112Lys), +/− heterozygous variant identified, +/+ wild type on both alleles, verified by PCR amplification and Sanger sequencing. B Sanger sequencing chromatogram of SPARCL1 exon 4 in affected individual III:8 demonstrating a heterozygous c.334G > A. C The functional motifs are as follows: signal peptide; FOLN (follistatin/osteonectin-like EGF domain); Kazal 1 (Kazal-type serine protease inhibitor domain); SPARC Ca bdg (secreted protein acidic and rich in cysteine Ca binding region). The disordered regions, parts of the protein lacking definition, are represented by light grey shading. Low-complexity regions are represented in blue shading. p.(Glu112Lys) is located in a disordered region of the protein. Domains are derived from data in Pfam. D Conservation of protein sequence across 14 species. E RNA-seq transcript expression of SPARCL1 in the different layers of the cornea. BLC basal limbal crypts, SLC superficial limbal crypts. Data were curated from bulk RNA-seq and presented as transcripts per million (TPM) [ , ].

Article Snippet: Sections were incubated in primary antibodies goat anti-SPARCL1 (AF2728, R&D Systems, Minneapolis, MN, USA, 1:50) and rabbit anti-DCN (LF-122, Kerafast, Boston, MA, USA, 1:200), at room temperature for 1.5 h then 4 °C overnight.

Techniques: Variant Assay, Amplification, Sequencing, Functional Assay, Protease Inhibitor, Binding Assay, Derivative Assay, RNA Sequencing Assay, Expressing

Journal: European Journal of Human Genetics

Article Title: Autosomal dominant stromal corneal dystrophy associated with a SPARCL1 missense variant

doi: 10.1038/s41431-024-01687-8

Figure Lengend Snippet: A , E Merge of DAPI and SPARCL1 channels show an upregulation of SPARCL1 in the epithelial layer of the affected tissue. B , F Merge DAPI and decorin channels. The downregulation of decorin is evident in the stroma of the affected tissue. C , G Merge of all three channels. Scale bars correspond to 50 μm. D , H Magnification box of ( C ) and ( G ), respectively, scale bars correspond to 5 μm, perinuclear co-localisation of SPARCL1 and decorin is observed in affected tissue.

Article Snippet: Sections were incubated in primary antibodies goat anti-SPARCL1 (AF2728, R&D Systems, Minneapolis, MN, USA, 1:50) and rabbit anti-DCN (LF-122, Kerafast, Boston, MA, USA, 1:200), at room temperature for 1.5 h then 4 °C overnight.

Techniques:

Journal: PLoS ONE

Article Title: Secreted protein acidic and rich in cysteine (SPARC) knockout mice have greater outflow facility

doi: 10.1371/journal.pone.0241294

Figure Lengend Snippet: Antibodies used for immunoblotting and immunofluorescence.

Article Snippet: Matricellular , Hevin , Proteintech , Rabbit , 1:100 , Goat anti-rabbit 594 , Molecular Probes , 1:200.

Techniques: Western Blot, Immunofluorescence

Journal: PLoS ONE

Article Title: Secreted protein acidic and rich in cysteine (SPARC) knockout mice have greater outflow facility

doi: 10.1371/journal.pone.0241294

Figure Lengend Snippet: ECM and matricellular protein percent change in SPARC -/- mice compared to WT mice. Percent change in TM tissue was determined through IHC. Percent change in MTM culture was determined through immunoblotting.

Article Snippet: Matricellular , Hevin , Proteintech , Rabbit , 1:100 , Goat anti-rabbit 594 , Molecular Probes , 1:200.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Secreted protein acidic and rich in cysteine (SPARC) knockout mice have greater outflow facility

doi: 10.1371/journal.pone.0241294

Figure Lengend Snippet: Fluorescence intensities of (A) matricellular and (B) ECM proteins and PAI-1 in WT and SPARC -/- mice. WT mice had greater intensities of collagen types IV and VI, fibronectin, laminin, PAI-1, and tenascin-C (***, p < 0.05). Error bars represent standard deviations.

Article Snippet: Matricellular , Hevin , Proteintech , Rabbit , 1:100 , Goat anti-rabbit 594 , Molecular Probes , 1:200.

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Secreted protein acidic and rich in cysteine (SPARC) knockout mice have greater outflow facility

doi: 10.1371/journal.pone.0241294

Figure Lengend Snippet: SC: Schlemm’s canal. TM: trabecular meshwork. C: cornea. Collagen types IV and VI, fibronectin, laminin, PAI-1, and TNC were all significantly decreased in SPARC -/- mice. Other ECM and matricellular proteins exhibited no significant differences in intensity between WT and SPARC -/- mice; these images have been omitted in the interest of space. Not shown: SPARC labeling was negative in all SPARC -/- mice. Scale bars: 20 μm.

Article Snippet: Matricellular , Hevin , Proteintech , Rabbit , 1:100 , Goat anti-rabbit 594 , Molecular Probes , 1:200.

Techniques: Labeling