Journal: Oncotarget

Article Title: ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis

doi: 10.18632/oncotarget.19018

Figure Lengend Snippet: A. ANGPTL4 protein expression level in UCLA-SO-M12, UCLA-SO-M16 and DP-0574-Me cutaneous (CUT) and melanoma brain metastasizing (MBM) variants of first and second IC inoculation cycle was analyzed using Western blotting. The obtained values were normalized to β-Tubulin. The bars represent the relative expression of ANGPTL4 (normalized to RS9), compared to control, untreated cells + SD obtained in one measurement in at least three independent experiments. * P < 0.05. B. , C. ANGPTL4 expression in paired samples of primary melanoma (PRM), melanoma lymph node metastasis (LNM), and melanoma brain metastasis (MBM) derived from melanoma patients. (B) Representative IHC staining with anti-ANGPTL4 Ab for PRM, LNM and MBM specimens. Black bars indicate 100µm. The insets show a magnification of the melanoma lesions. Black arrowheads indicate ANGPTL4 positive melanoma cells. Yellow bars indicate 20µm. (C) Box plot comparing H score for PRM, LNM and MBM. * P < 0.01, ** P < 0.005, *** P < 0.0005. D. Melanoma cells were incubated with 5ng/ml TGFβ1 for 4 hrs. Following stimulation, RT-qPCR analysis was performed to determine the mRNA expression level of ANGPTL4. The bars represent the relative expression of ANGPTL4 (normalized to RS9), compared to control, untreated cells + SD obtained in one measurement in at least three independent experiments. * P < 0.05. E. Brains of BALB/c mice were harvested, and BDF were prepared after 24 hrs (see Materials and Methods) and added to melanoma cells for 24 hrs at 37°C. Melanoma cells treated with 0.5% BSA supplemented RPMI-1640 served as control. Following stimulation, RT-qPCR analysis was performed to determine the mRNA expression level of ANGPTL4. The bars represent the relative expression of ANGPTL4 (normalized to RS9), compared to control, untreated cells + SEM obtained in one measurement in at least three independent experiments. * P < 0.05. F. , G. CM of microglia (MG), astrocytes (HA) and BEC was collected, and added to YDFR.CB3 (F) and DP.CB2 (G) melanoma cells for 24 hrs at 37°C. Melanoma cells treated with starvation medium served as control. Following stimulation, RT-qPCR analysis was performed to determine the mRNA expression level of ANGPTL4. The bars represent the relative expression of ANGPTL4 (normalized to RS9), compared to control, untreated cells + SEM obtained in one measurement in at least three independent experiments. * P < 0.05, ** P < 0.005.

Article Snippet: The sections were incubated overnight with the anti-ANGPTL4 rabbit polyclonal Ab (Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:100 in a 4°C humidified chamber.

Techniques: Expressing, Western Blot, Control, Derivative Assay, Immunohistochemistry, Incubation, Quantitative RT-PCR

Journal: Oncotarget

Article Title: ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis

doi: 10.18632/oncotarget.19018

Figure Lengend Snippet: A. - C. Cutaneous (CUT) and melanoma brain metastasizing (MBM) variants were transduced with an ANGPTL4 cDNA-containing construct (ANGPTL4 hi ) or with the backbone construct pQCXIP (CON pQC ). MBM cells were transduced with a mixture of 4 different shANGPTL4-containing constructs (ANGPTL4 lo ), or with the control construct (CON sh ). The efficacy of ANGPTL4 over-expression or down-regulation was verified: (A) RT-qPCR analysis was performed to determine the mRNA expression level of ANGPTL4. The bars represent the relative ANGPTL4 expression (normalized to RS9) in ANGPTL4 hi or ANGPTL4 lo cells compared to control cells + SEM obtained in one measurement in at least three independent experiments. * P < 0.05. (B, C) Supernatants of the transduced cells were subjected to Western blot analysis. ANGPTL4 was detected by specific Abs. The bars represent the relative ANGPTL4 expression in ANGPTL4 hi or ANGPTL4 lo cells compared to control cells + SEM obtained in one measurement in two-three independent experiments. * P < 0.05. Representative blots are shown. Ponceau staining was used for loading control. D. , E. Melanoma cells were allowed to migrate through collagen coated transwells for 24 hrs. The migrated cells were fixed and counted. (D) Representative images are presented (X10 magnification). (E) The bars represent the average number of migrating cells per field in three independent experiments performed in triplicates + SEM. * P < 0.05. F. BEC were cultured for 24 hrs to form a confluent monolayer and stimulated with 100 units/ml TNFα and IFNγ for additional 24 hrs. mCherry- or GFP-expressing melanoma cells were added and allowed to adhere for 30 min at 37°C. The fluorescence signal of labeled cells was measured before and after removal of non-adherent cells. The bars represent the average % adherent cells (normalized to control cells) + SEM in at least three independent experiments. Six replicates were performed in each experiment. * P < 0.05. G. - I. BEC were cultured for 48 hrs on the upper side of the apical chamber of transwell inserts to form a confluent monolayer. Astrocytes were cultured in 24-well plates for 24 hrs and starved for additional 24 hrs to allow the secretion of soluble factors. mCherry-expressing melanoma cells were added onto the BEC monolayer and allowed to migrate for 24 hrs towards the astrocytes (G). The migrated cells were fixed and counted. (H) Representative images are presented (X10 magnification). (I) The bars represent the average number of migrating cells per field (normalized to control cells) in at least three independent experiments performed in triplicates + SEM. * P < 0.05.

Article Snippet: The sections were incubated overnight with the anti-ANGPTL4 rabbit polyclonal Ab (Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:100 in a 4°C humidified chamber.

Techniques: Transduction, Construct, Control, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Staining, Cell Culture, Fluorescence, Labeling

Journal: Oncotarget

Article Title: ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis

doi: 10.18632/oncotarget.19018

Figure Lengend Snippet: A. - C. Melanoma cells were cultured for 24 hrs, then starved for additional 24 hrs, to allow secretion of melanoma-soluble factors. CM was collected and added to BEC. Cell viability of BEC grown with CM of cutaneous CON pQC and ANGPTL4 hi melanoma cells (A) or MBM CON pQC and ANGPTL4 hi melanoma cells (B) was monitored after 24, 48 and 120 hrs. Cell viability of BEC grown with CM of MBM CON sh and ANGPTL4 lo melanoma cells (C) was monitored after 72 hrs. Viability was determined using XTT-based assay. Absorbance at 450 nm was determined for each well, and subtraction of nonspecific readings (measured at 630 nm) was calculated. The bars represent the average viability of the BEC grown with ANGPTL4 hi or ANGPTL4 lo melanoma CM relative to those grown in control melanoma CM + SD in three independent experiments. Six replicates were performed in each experiment. * P < 0.05, ** P < 0.05. D. - G. Melanoma cells were seeded for 24 hrs and then cultured in serum-free media for additional 24 hrs. CM were collected and subjected to gelatin and collagen zymography. (D, F) Images of one representative experiment out of three are presented. A ~66 kDa band indicating the active form of MMP-2 was observed. (E, G) The bars represent the average MMP-2 activity (pixel density) as measured by densitometry, normalized to control cells + SD in one measurement in three independent experiments. * P < 0.05, ** P < 0.0.1, *** P < 0.005. H. , I. BECs were treated with melanoma CM for 24 hours. Then, CLDN1 expression was analyzed using RT-qPCR and flow cytometry. (H) The bars represent the relative CLDN1 mRNA expression (normalized to RS9) compared to control cells + SD obtained in one measurement in least three independent experiments. * P < 0.05. (I) BEC were trypsinized and analyzed for CLDN1 expression using flow cytometry. Dot plot histogram comparing % CLDN1 positive BEC. Each dot represents the value obtained in a single independent experiment. The line represents the average value in each variant. ** P < 0.01.

Article Snippet: The sections were incubated overnight with the anti-ANGPTL4 rabbit polyclonal Ab (Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:100 in a 4°C humidified chamber.

Techniques: Cell Culture, XTT Assay, Control, Zymography, Activity Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Variant Assay

Journal: Oncotarget

Article Title: ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis

doi: 10.18632/oncotarget.19018

Figure Lengend Snippet: A. Volume of cutaneous tumors following subdermal implantation of CON pQC vs. ANGPTL4 hi cutaneous cells. Tumor dimensions were measured using a caliper and volume was obtained as described in Materials and Methods. The average tumor volume + SEM is presented. * P < 0.05. B. Mice were weighed weekly following melanoma inoculation. The averege mouse weight + SD is presented. * P < 0.05. C. Melanoma cells were treated for 72 hrs with mouse BDF. Then, cells were trypsinized and cell death was determined by measuring DAPI incorporation. The bars represent the relative cell death of BDF treated cells compared to their controls + SEM obtained in one measurement in three independent experiments. * P < 0.05.

Article Snippet: The sections were incubated overnight with the anti-ANGPTL4 rabbit polyclonal Ab (Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:100 in a 4°C humidified chamber.

Techniques:

Journal: Oncotarget

Article Title: ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis

doi: 10.18632/oncotarget.19018

Figure Lengend Snippet: The expression of 305 proteins was examined using RPPA analysis in the ANGPTL4 hi and ANGPTL4 lo cells vs. their corresponding control cells. A. , B. , C. Comparisons were done for each pair of cells: (A) cutaneous ANGPTL4 hi vs. CON pQC cells, (B) brain metastasizing ANGPTL4 hi vs. CON pQC cells and (C) brain metastasizing ANGPTL4 lo vs. CON sh cells. The tables show the normalized expression of differentially expressed proteins with FC < -1.25 or FC>1.25. D. , E. Venn diagrams were used to compare lists of differentially expressed proteins. (D) Proteins differentially expressed in both comparisons obtained in (A) and (B). (E) Proteins differentially expressed in both comparisons obtained in (B) and (C). F. Protein-protein interactions of differentially expressed proteins with FC < -1.5 or FC>1.5 from all three comparisons. Circled in blue are proteins from comparison (A), circled in orange are proteins from comparison (B), and circled in green are proteins from comparison (C).

Article Snippet: The sections were incubated overnight with the anti-ANGPTL4 rabbit polyclonal Ab (Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:100 in a 4°C humidified chamber.

Techniques: Expressing, Control, Protein-Protein interactions, Comparison

Journal: Oncotarget

Article Title: ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis

doi: 10.18632/oncotarget.19018

Figure Lengend Snippet: A soluble factor in the microenvironment of the primary tumor transforming growth factor β1 (TGFβ1) induces the expression of ANGPTL4 in primary melanoma tumor cells. ANGPTL4 enhances their ability to migrate through extracellular matrix (ECM) components and to adhere and invade brain vasculature, for example by down-regulating the expression of cell-cell adhesion tight junction (TJ) molecules such as claudin-1 (CLDN1). Once arriving the brain, brain-derived soluble factors secreted by microglia, brain endothelial cells (BEC) and astrocytes, induce ANGPTL4 expression by brain metastasizing cells, what contributes to different phenotypes, such as resistance against brain-derived cytotoxic factors, enhancement of BEC growth and induction of angiogenesis-related genes such as angiopoietin 1 (ANG1) (data not shown) in BEC subjected to factors released from brain metastasizing cells, expressing high ANGPTL4 levels.

Article Snippet: The sections were incubated overnight with the anti-ANGPTL4 rabbit polyclonal Ab (Proteintech Group, Inc, Rosemont, IL, USA) at a dilution of 1:100 in a 4°C humidified chamber.

Techniques: Expressing, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins

doi: 10.1523/JNEUROSCI.1153-17.2017

Figure Lengend Snippet: Expression and distribution of cadherin-10 in cortex. A, In situ hybridization of cadherin-10, reproduced from the Allen Brain Atlas database showing high expression of cadherin-10 in cerebral cortex and CA1 region of hippocampus. Scale bars, 1 mm. B, Representative Western blot (WB) of whole brain lysate, probed with cadherin-10 antibody (n = 4 independent experiments). C, Representative WB of lysates from HEK293 cells expressing myccadherin-10, GFPN-cadherin, or untransfected control (UT). Blots are probed with antibodies to cadherin-10 or GFP (n = 3 independent experiments). D, Light microscopy image of layer 2/3 of cerebral cortex immunostained for cadherin-10. Scale bar, 10 μm. E, Enlargement of the boxed region in D, immunostained for cadherin-10 (green) and synaptophysin (red); closely apposed puncta are marked with solid white circles and overlapping puncta with dotted circles. Scale bar, 2 μm. F, Quantification of cadherin-10 and synaptophysin (Syn.) colocalization showing 37 ± 5% of cadherin-10 clusters were positive (pos.) for synaptophysin and 40 ± 2% synaptophysin clusters were positive for cadherin-10.

Article Snippet: The following antibodies were purchased: mouse anti-PSD-95 monoclonal (RRID: AB_2292909 ; Neuromab), mouse anti-GFP monoclonal (RRID: AB_1587098 ; Millipore), chicken anti-GFP polyclonal (RRID: AB_300798 ; Abcam), mouse anti-myc monoclonal (University of Iowa Hybridoma Bank and RRID: AB_627266 ; Santa Cruz Biotechnology), rabbit anti-cadherin-10 polyclonal (T2, to the C terminus, RRID: AB_2275951 ; Santa Cruz Biotechnology), mouse anti-turboGFP (RRID: AB_2622256 ; Origene), rabbit anti-α2 GABA A R (RRID: AB_2108839 ; Synaptic Systems), mouse anti-gephyrin (RRID: AB_887716 for immunocytochemistry, RRID: AB_887719 for Western blot; Synaptic Systems), anti-synaptophysin antibody (RRID: AB_477523 ; Sigma-Aldrich), rabbit anti-GluA1 (RRID: AB_1977216 ; Millipore), mouse anti-β3-GABA A R (RRID: AB_2109585 ; Neuromab), mouse anti-NL2 (RRID: AB_993011 ; Synaptic Systems), mouse anti-NL1 (RRID: AB_2235964 ; Neuromab), mouse anti-Caspr2 (RRID: AB_2245198 ; Neuromab), anti-N-cadherin (RRID: AB_398236 ; BD Biosciences).

Techniques: Expressing, In Situ Hybridization, Western Blot, Light Microscopy

Journal: The Journal of Neuroscience

Article Title: Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins

doi: 10.1523/JNEUROSCI.1153-17.2017

Figure Lengend Snippet: Cadherin-10 is localized to excitatory and inhibitory presynaptic and postsynaptic sites. A, Immunogold-EM micrographs of excitatory and inhibitory synapses from layer 2/3 of neocortex. A glutamatergic terminal is colorized in pink, a postsynaptic spine in green, a GABAergic terminal in blue, and opposing postsynaptic dendrites in yellow. Large dots are gold particles coding for cadherin-10, and small dots code for GABA. Arrowheads point to cadherin-10 immunoreactivity. Scale bars, 200 nm. B, C, Quantitative analysis of cadherin-10 gold particle position relative to the postsynaptic plasma membrane in the perpendicular axis (B) and lateral position tangentially along the plasma membrane (C; position 0, central; position 1, perisynaptic; position 2, extrasynaptic; 100 synapses from 4 rats). D, Confocal image of DIV 21 cortical dendrite expressing GFP and immunostained with antibodies to cadherin-10. Scale bar, 5 μm. Arrowheads point to cadherin-10 in spines, and open arrowheads point to cadherin-10 in the dendritic shaft. E, Confocal images of DIV 21 cortical dendrites immunostained with antibodies to cadherin-10 PSD-95 (left) and gephyrin (right); bar graphs quantify the percentage of cadherin-10 (Cad-10) clusters that colocalize with synaptic marker clusters. Scale bars, 5 μm. F, Confocal image of cortical neuron expressing myccadherin-10 and GFP. Filled arrowheads point to cadherin-10 puncta in spines, and open arrowheads point to cadherin-10 in the dendritic shaft. Scale bar, 5 μm. G, Confocal image of cortical neuron expressing myccadherin-10 and immunostained with antibodies to vesicular GABA transporter to identify GABAergic terminals.

Article Snippet: The following antibodies were purchased: mouse anti-PSD-95 monoclonal (RRID: AB_2292909 ; Neuromab), mouse anti-GFP monoclonal (RRID: AB_1587098 ; Millipore), chicken anti-GFP polyclonal (RRID: AB_300798 ; Abcam), mouse anti-myc monoclonal (University of Iowa Hybridoma Bank and RRID: AB_627266 ; Santa Cruz Biotechnology), rabbit anti-cadherin-10 polyclonal (T2, to the C terminus, RRID: AB_2275951 ; Santa Cruz Biotechnology), mouse anti-turboGFP (RRID: AB_2622256 ; Origene), rabbit anti-α2 GABA A R (RRID: AB_2108839 ; Synaptic Systems), mouse anti-gephyrin (RRID: AB_887716 for immunocytochemistry, RRID: AB_887719 for Western blot; Synaptic Systems), anti-synaptophysin antibody (RRID: AB_477523 ; Sigma-Aldrich), rabbit anti-GluA1 (RRID: AB_1977216 ; Millipore), mouse anti-β3-GABA A R (RRID: AB_2109585 ; Neuromab), mouse anti-NL2 (RRID: AB_993011 ; Synaptic Systems), mouse anti-NL1 (RRID: AB_2235964 ; Neuromab), mouse anti-Caspr2 (RRID: AB_2245198 ; Neuromab), anti-N-cadherin (RRID: AB_398236 ; BD Biosciences).

Techniques: Expressing, Marker

Journal: The Journal of Neuroscience

Article Title: Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins

doi: 10.1523/JNEUROSCI.1153-17.2017

Figure Lengend Snippet: Cadherin-10 forms nanoscale puncta at cortical synapses A, SIM images of cortical dendrites immunostained for cadherin-10 and PSD-95 or gephyrin (maximum projections of z-stacks). Filled arrowheads point to cadherin-10-positive clusters, and open arrowheads point to cadherin-10-negative clusters. Scale bar, 2 μm. B, High-magnification views of cadherin-10 puncta at PSD-95 and gephyrin clusters. White lines show the path of line scans represented in the graphs below the images. C, D, Confocal image of a DIV 24 cortical neuron immunostained for cadherin-10 and PSD-95 (scale bar, 5 μm; maximum projection of z-stack). The boxed region is shown at higher magnification below. White lines (a′ and b′) produced line scans in D showing similar cluster diameter for cadherin-10 and PSD-95 when imaged by confocal microscopy. E, Top, High-magnification SIM image showing the small cadherin-10 cluster lies adjacent to the large gephyrin cluster. To analyze the cadherin-10 puncta diameter, we generated a line scan (right, red line). Bottom, Gaussian fit of pixel-intensity profile across the cadherin-10 cluster. FWHM of profile was used to determine cluster diameter. F, H, Frequency distributions of PSD-95 (F), gephyrin (G), and cadherin-10 (H) cluster diameter. I, Mean diameter of PSD-95, gephyrin, and cadherin-10 clusters; PSD-95, 0.409 ± 0.009 μm; gephyrin, 0.335 ± 0.008 μm; cadherin-10, 0.196 ± 0.003 μm; ****p = 0.0001, Kruskal–Wallis test; n = 132–195 PSDs. J, Percentages of cadherin-10-positive PSD-95 and gephyrin clusters (PSD-95, 80.6 ± 5.4%; gephyrin, 49.4 ± 6.7%; *p = 0.012, t test; n = 4–5 neurons).

Article Snippet: The following antibodies were purchased: mouse anti-PSD-95 monoclonal (RRID: AB_2292909 ; Neuromab), mouse anti-GFP monoclonal (RRID: AB_1587098 ; Millipore), chicken anti-GFP polyclonal (RRID: AB_300798 ; Abcam), mouse anti-myc monoclonal (University of Iowa Hybridoma Bank and RRID: AB_627266 ; Santa Cruz Biotechnology), rabbit anti-cadherin-10 polyclonal (T2, to the C terminus, RRID: AB_2275951 ; Santa Cruz Biotechnology), mouse anti-turboGFP (RRID: AB_2622256 ; Origene), rabbit anti-α2 GABA A R (RRID: AB_2108839 ; Synaptic Systems), mouse anti-gephyrin (RRID: AB_887716 for immunocytochemistry, RRID: AB_887719 for Western blot; Synaptic Systems), anti-synaptophysin antibody (RRID: AB_477523 ; Sigma-Aldrich), rabbit anti-GluA1 (RRID: AB_1977216 ; Millipore), mouse anti-β3-GABA A R (RRID: AB_2109585 ; Neuromab), mouse anti-NL2 (RRID: AB_993011 ; Synaptic Systems), mouse anti-NL1 (RRID: AB_2235964 ; Neuromab), mouse anti-Caspr2 (RRID: AB_2245198 ; Neuromab), anti-N-cadherin (RRID: AB_398236 ; BD Biosciences).

Techniques: Produced, Confocal Microscopy, Generated

Journal: The Journal of Neuroscience

Article Title: Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins

doi: 10.1523/JNEUROSCI.1153-17.2017

Figure Lengend Snippet: Cadherin-10 knockdown reduces E/I synapse ratio in dendrites of cortical neurons A, Western blot (WB) analysis of lysates from HEK293 cells expressing myccadherin-10 alone, or with GFP-expressing control or candidate RNAi constructs. The red box highlights effects of RNAi 2. B, Quantification of cadherin-10 RNAi knockdown in HEK293 cells. RNAi construct 2, which proved very effective, had no effect on N-cadherin expression and was used for all experiments (****p < 0.0001, 1-way ANOVA; n = 3 independent experiments). C, Maximum projections of confocal z-stacks showing cortical neurons expressing control or cadherin-10 RNAi and immunostained with antibodies to cadherin-10. Scale bar, 20 μm. Arrowheads point to cadherin-10 expression in soma and dendrites of neurons. Bar graphs show quantification of cadherin-10 in the soma and dendrites of control and knock-down neurons (**p = 0.0019; ***p = 0.0009; n = 6–9 neurons). D, Maximum projections of confocal z-stacks showing control, cadherin-10 knockdown, or knockdown and rescue expressing DIV 24 cortical neurons; neurons also express GFP to outline the dendrite. Scale bar, 5 μm. E, Quantification of spine area, width, length, and density (area, **p = 0.0043; width,**p = 0.0037; length,*p = 0.0424; density, p > 0.05; n = 19–20 neurons). F, Maximum projections of confocal z-stacks showing control, cadherin-10 knockdown, or knockdown and rescue of GFP-expressing DIV 24 cortical neurons (green) labeled with antibodies to α2-GABAAR clusters (red). Scale bar, 5 μm. G. Quantification of α2-GABAAR cluster area and density (area, *p = 0.0258; density, p > 0.05; n = 17–23 neurons). H, Quantification of ratio of spine area to GABAAR cluster area in the same dendrite. The bar graph shows the E/I ratio is reduced from 4.9 ± 0.5 to 3.0 ± 1.3 in cadherin-10 knock-down neurons (E/I: *p = 0.010, n = 10–12 neurons).

Article Snippet: The following antibodies were purchased: mouse anti-PSD-95 monoclonal (RRID: AB_2292909 ; Neuromab), mouse anti-GFP monoclonal (RRID: AB_1587098 ; Millipore), chicken anti-GFP polyclonal (RRID: AB_300798 ; Abcam), mouse anti-myc monoclonal (University of Iowa Hybridoma Bank and RRID: AB_627266 ; Santa Cruz Biotechnology), rabbit anti-cadherin-10 polyclonal (T2, to the C terminus, RRID: AB_2275951 ; Santa Cruz Biotechnology), mouse anti-turboGFP (RRID: AB_2622256 ; Origene), rabbit anti-α2 GABA A R (RRID: AB_2108839 ; Synaptic Systems), mouse anti-gephyrin (RRID: AB_887716 for immunocytochemistry, RRID: AB_887719 for Western blot; Synaptic Systems), anti-synaptophysin antibody (RRID: AB_477523 ; Sigma-Aldrich), rabbit anti-GluA1 (RRID: AB_1977216 ; Millipore), mouse anti-β3-GABA A R (RRID: AB_2109585 ; Neuromab), mouse anti-NL2 (RRID: AB_993011 ; Synaptic Systems), mouse anti-NL1 (RRID: AB_2235964 ; Neuromab), mouse anti-Caspr2 (RRID: AB_2245198 ; Neuromab), anti-N-cadherin (RRID: AB_398236 ; BD Biosciences).

Techniques: Western Blot, Expressing, Construct, Labeling

Journal: The Journal of Neuroscience

Article Title: Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins

doi: 10.1523/JNEUROSCI.1153-17.2017

Figure Lengend Snippet: Cadherin-10 loss reciprocally modulates excitatory and inhibitory synaptic strength. A, Representative AMPA receptor-mediated mEPSC and GABAAR-mediated mIPSC traces from control or cadherin-10 knock-down DIV 24 cortical neurons. B, Bar graphs of mEPSC and mIPSC amplitudes showing mEPSCs are reduced from 28.7 ± 3.8 pA to 20.3 ± 1.0 pA, and mIPSCs are increased from 24.0 ± 1.9 pA to 29.6 ± 1.7 pA (mEPSCs, *p = 0.040; mIPSCs, *p = 0.034; t tests; n = 14–15 neurons). C, Cumulative frequency graphs of mEPSC and mIPSC amplitudes. D, Bar graphs of mEPSC and mIPSC frequencies showing no significant difference (mEPSCs, p = 0.282; mIPSCs, p = 0.326; t tests; n = 12–13 neurons).

Article Snippet: The following antibodies were purchased: mouse anti-PSD-95 monoclonal (RRID: AB_2292909 ; Neuromab), mouse anti-GFP monoclonal (RRID: AB_1587098 ; Millipore), chicken anti-GFP polyclonal (RRID: AB_300798 ; Abcam), mouse anti-myc monoclonal (University of Iowa Hybridoma Bank and RRID: AB_627266 ; Santa Cruz Biotechnology), rabbit anti-cadherin-10 polyclonal (T2, to the C terminus, RRID: AB_2275951 ; Santa Cruz Biotechnology), mouse anti-turboGFP (RRID: AB_2622256 ; Origene), rabbit anti-α2 GABA A R (RRID: AB_2108839 ; Synaptic Systems), mouse anti-gephyrin (RRID: AB_887716 for immunocytochemistry, RRID: AB_887719 for Western blot; Synaptic Systems), anti-synaptophysin antibody (RRID: AB_477523 ; Sigma-Aldrich), rabbit anti-GluA1 (RRID: AB_1977216 ; Millipore), mouse anti-β3-GABA A R (RRID: AB_2109585 ; Neuromab), mouse anti-NL2 (RRID: AB_993011 ; Synaptic Systems), mouse anti-NL1 (RRID: AB_2235964 ; Neuromab), mouse anti-Caspr2 (RRID: AB_2245198 ; Neuromab), anti-N-cadherin (RRID: AB_398236 ; BD Biosciences).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins

doi: 10.1523/JNEUROSCI.1153-17.2017

Figure Lengend Snippet: Cadherin-10 associates with PSD-95 and gephyrin A, Representative Western blot of cadherin-10 coIP from cultured cortical neurons (DIV 22) probed with synaptic proteins. A strong PSD-95 band and weak N-cadherin band are visible in the immunoprecipitation lane (n = 4 independent experiments). B, Representative Western blot of cadherin-10 coIP from rat cortical homogenate, showing interaction with PSD-95 but not gephyrin (n = 3 independent experiments). C, Representative Western blot of gephyrin coIP from rat cortical homogenate, showing interaction with cadherin-10 (n = 3 independent experiments). D, Representative Western blot of cortical membrane fractions. S1 and S2, crude homogenate; S3, membrane-associated protein; S5, membrane protein; S6, PSD protein. Cadherin-10 shows a strong band in the membrane-associated fraction, whereas a comparable band is barely visible for N-cadherin (n = 3 independent experiments). IP, Immunoprecipitation; MW, molecular weight.

Article Snippet: The following antibodies were purchased: mouse anti-PSD-95 monoclonal (RRID: AB_2292909 ; Neuromab), mouse anti-GFP monoclonal (RRID: AB_1587098 ; Millipore), chicken anti-GFP polyclonal (RRID: AB_300798 ; Abcam), mouse anti-myc monoclonal (University of Iowa Hybridoma Bank and RRID: AB_627266 ; Santa Cruz Biotechnology), rabbit anti-cadherin-10 polyclonal (T2, to the C terminus, RRID: AB_2275951 ; Santa Cruz Biotechnology), mouse anti-turboGFP (RRID: AB_2622256 ; Origene), rabbit anti-α2 GABA A R (RRID: AB_2108839 ; Synaptic Systems), mouse anti-gephyrin (RRID: AB_887716 for immunocytochemistry, RRID: AB_887719 for Western blot; Synaptic Systems), anti-synaptophysin antibody (RRID: AB_477523 ; Sigma-Aldrich), rabbit anti-GluA1 (RRID: AB_1977216 ; Millipore), mouse anti-β3-GABA A R (RRID: AB_2109585 ; Neuromab), mouse anti-NL2 (RRID: AB_993011 ; Synaptic Systems), mouse anti-NL1 (RRID: AB_2235964 ; Neuromab), mouse anti-Caspr2 (RRID: AB_2245198 ; Neuromab), anti-N-cadherin (RRID: AB_398236 ; BD Biosciences).

Techniques: Western Blot, Cell Culture, Immunoprecipitation, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins

doi: 10.1523/JNEUROSCI.1153-17.2017

Figure Lengend Snippet: Nanoscale distribution of cadherin-10 puncta impacts synaptic scaffold dimensions A, Mean number of cadherin-10 puncta per PSD-95- and gephyrin-positive synapse. Gephyrin PSDs have 50.6% fewer cadherin-10 puncta compared with PSD-95. Data are also shown as a frequency distribution histogram (****p < 0.0001, n = 170–195 PSDs). Right, cadherin-10 puncta number at PSD-95 and gephyrin PSDs, normalized to PSD diameter (****p < 0.0001, n = 170–195 PSDs). B, SIM images show examples of different cadherin-10 puncta distributions. Scale bar, 0.2 μm; maximum projections of z-stacks. C, Bar graphs showing percentage of PSD-95 and gephyrin synapses with different distributions of cadherin-10 puncta; the distribution of cadherin-10 puncta was similar at both PSDs (2-way ANOVA, p > 0.05; n = 4–5 neurons). Forty-nine percent of gephyrin PSDs had no cadherin-10 compared with 20% of PSD-95 PSDs (Bonferroni's post hoc test, **p < 0.001; n = 4–5 neurons). D, PSD diameter correlates with subsynaptic distribution of cadherin-10 (***p = 0.0005; *p = 0.0132; 1-way ANOVA; n = 170–195 PSDs). E. SIM images of different numbers of cadherin-10 puncta at PSD-95- and gephyrin-positive synapses. Scale bar, 0.2 μm; maximum projections of z-stack. F, Correlation between PSD-95 or gephyrin diameter and number of cadherin-10 puncta (n = 170–195 PSDs, 4–5 neurons per condition). G, Presence of cadherin-10 puncta correlates significantly with PSD-95 diameter but not gephyrin diameter (0.365 ± 0.01 μm to 0.419 ± 0.01 μm; PSD-95, *p = 0.015; gephyrin, p = 0.197; Mann–Whitney test; n = 170–195 PSDs; 4–5 neurons per condition). H, Maximum projections of confocal z-stacks showing PSD-95 (red) in control and cadherin-10 knock-down neurons. Scale bar, 5 μm. I, Total dendritic PSD-95 intensity and total cluster density in control and knock-down neurons. Both the total dendritic PSD-95 intensity and cluster density are reduced in knock-down neurons compared with control (total intensity: 416.9 ± 23.4 to 323 ± 16.3.3 mean gray values, **p = 0.004; density: 9.2 ± 1.2 to 6.4 ± 0.8 PSD-95 puncta/10 μm, *p = 0.035, Mann–Whitney tests; n = 10–11 neurons). J, Schematic diagram summarizes the effects of cadherin-10 knockdown on excitatory and inhibitory synapses. Cadherin-10 puncta are more abundant at excitatory synapses than inhibitory synapses. Cadherin-10 knockdown suppresses excitatory synaptic transmission and enhances inhibitory synaptic transmission, leading to a decrease in the dendritic E/I ratio.

Article Snippet: The following antibodies were purchased: mouse anti-PSD-95 monoclonal (RRID: AB_2292909 ; Neuromab), mouse anti-GFP monoclonal (RRID: AB_1587098 ; Millipore), chicken anti-GFP polyclonal (RRID: AB_300798 ; Abcam), mouse anti-myc monoclonal (University of Iowa Hybridoma Bank and RRID: AB_627266 ; Santa Cruz Biotechnology), rabbit anti-cadherin-10 polyclonal (T2, to the C terminus, RRID: AB_2275951 ; Santa Cruz Biotechnology), mouse anti-turboGFP (RRID: AB_2622256 ; Origene), rabbit anti-α2 GABA A R (RRID: AB_2108839 ; Synaptic Systems), mouse anti-gephyrin (RRID: AB_887716 for immunocytochemistry, RRID: AB_887719 for Western blot; Synaptic Systems), anti-synaptophysin antibody (RRID: AB_477523 ; Sigma-Aldrich), rabbit anti-GluA1 (RRID: AB_1977216 ; Millipore), mouse anti-β3-GABA A R (RRID: AB_2109585 ; Neuromab), mouse anti-NL2 (RRID: AB_993011 ; Synaptic Systems), mouse anti-NL1 (RRID: AB_2235964 ; Neuromab), mouse anti-Caspr2 (RRID: AB_2245198 ; Neuromab), anti-N-cadherin (RRID: AB_398236 ; BD Biosciences).

Techniques: MANN-WHITNEY, Transmission Assay

Journal: Nature Communications

Article Title: Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction

doi: 10.1038/ncomms6066

Figure Lengend Snippet: ( a ) Surface NL2 derived from cultured hippocampal neurons of Pin1+/+ and Pin1−/− mice was isolated by biotinylation assay and detected by anti-NL2 antibody. No biotinylated neuronal cells were processed in parallel to evaluate unspecific NL2 binding. Western blot detecting glycophosphatidylinositol-anchored Flotilin was used as loading control ( n =4). Full images of western blots are in . ( b ) Typical examples of hippocampal neurons from Pin1+/+ and Pin1−/− immunolabeled for endogenous gephyrin (magenta), NL2 (green) and VGAT (blue) at DIV10. Enlarged boxed areas are shown aside to the corresponding full view image. Post-synaptic clustering is demonstrated by apposition of gephyrin/NL2 clusters to VGAT positive terminals on the merge window. Scale bars, 20 μm in full view images and 5 μm in enlarged panels. ( c ) Distribution histograms of NL2 cluster density (normalized to 100 μm 2 ), the average cluster size and intensity in Pin1+/+ and Pin1−/− hippocampal neurons. ( d ) Distribution histograms of the percentage of NL2 co-localizing with gephryin and the percentage of double labelled NL2/gephyrin puncta overlapping with the presynaptic marker VGAT. ( e ) Distribution histograms of gephyrin cluster density (normalized to 100 μm 2 ), the average cluster size and intensity (calculated as described in c ) in both mouse genotypes. The number of hippocampal neurons investigated in each experiments (three independent experiments) were as follows: n =10 for Pin1+/+, n =12 for Pin1−/−. For each neurons, at least five dendritic regions of interests were measured, mean values±s.d., ** P <0.001, *** P <0.0001, Student’s t -test).

Article Snippet: The following antibodies were used in immunohistochemistry and immunocitochemistry: anti-gephyrin Mab7a (Synaptic System Cat. No 147021), anti-VGAT rabbit or guinea pig (1:1,000, Synaptic System Cat. No 131004), anti-NL2 rabbit affinity purified (1:500, Synaptic system Cat, No 129203), guinea pig anti-GABA A γ2 subunit (1:2,000 (ref. )), biotinylated anti-guinea pig (1:200, Vector Laboratories, Cat No BA-7000).

Techniques: Derivative Assay, Cell Culture, Isolation, Cell Surface Biotinylation Assay, Binding Assay, Western Blot, Immunolabeling, Marker