08l100035 Search Results


mms 101p  (Valiant Co Ltd)
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Valiant Co Ltd mms 101p
Mms 101p, supplied by Valiant Co Ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse anti ha-tag mab  (Valiant Co Ltd)
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Valiant Co Ltd mouse anti ha-tag mab
Mouse Anti Ha Tag Mab, supplied by Valiant Co Ltd, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit gfp  (Valiant Co Ltd)
86
Valiant Co Ltd rabbit gfp
(a, b) Lysates from COS-7 cells transfected as shown at top were analyzed by IP and IB for proteins as indicated. For (b), densitometry of Plk2 associated <t>with</t> <t>NSF</t> plus GluA2: 3.45±0.34 (arbitrary units); minus GluA2: 1.57±0.15; p=0.03. (c) Lysates from cultured hippocampal neurons, infected for 18–20 h with Sindbis virus expressing Plk2 or <t>GFP,</t> were subjected to IP and IB as indicated. (d) Cultured hippocampal neurons were treated with PTX or vehicle (Veh.) for 24 h, and cell lysates used for IP and IB as indicated. (e) Lysates from cultured hippocampal neurons, infected for 18-20 h with Sind-Plk2 or -GFP, were subjected to IP and IB as indicated. Full-length blots are presented in . All MW in kDa. IN, Input, 5% lysate used for IPs. (f) Lysates of cultured hippocampal neurons infected with Sind-GFP or -Plk2 for 18–20 h were immunoblotted as indicated. Densitometry of phospho-ser880 with GFP: 0.25±0.09 (arbitrary units); with Plk2: 0.89±0.16; p=0.008. (g) COS-7 cells were transfected as shown at top, and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (sGluA2). (h) Quantification of data in (g); surface GluA2 was normalized to total GluA2, then plotted as percent of control. (i) HEK cells were transfected as indicated across top row. Cells were immunostained for sGluA2 (top row, red) and total GluA2 (tGluA2, middle row, green); overlay is shown in yellow (bottom row). Scale bar, 10 μm. (j) Quantification of surface to total GluA2 ratio from (i). **p<0.01; ***p<0.001.
Rabbit Gfp, supplied by Valiant Co Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit gfp - by Bioz Stars, 2026-02
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anti myc tag  (Valiant Co Ltd)
86
Valiant Co Ltd anti myc tag
(a, b) Lysates from COS-7 cells transfected as shown at top were analyzed by IP and IB for proteins as indicated. For (b), densitometry of Plk2 associated <t>with</t> <t>NSF</t> plus GluA2: 3.45±0.34 (arbitrary units); minus GluA2: 1.57±0.15; p=0.03. (c) Lysates from cultured hippocampal neurons, infected for 18–20 h with Sindbis virus expressing Plk2 or <t>GFP,</t> were subjected to IP and IB as indicated. (d) Cultured hippocampal neurons were treated with PTX or vehicle (Veh.) for 24 h, and cell lysates used for IP and IB as indicated. (e) Lysates from cultured hippocampal neurons, infected for 18-20 h with Sind-Plk2 or -GFP, were subjected to IP and IB as indicated. Full-length blots are presented in . All MW in kDa. IN, Input, 5% lysate used for IPs. (f) Lysates of cultured hippocampal neurons infected with Sind-GFP or -Plk2 for 18–20 h were immunoblotted as indicated. Densitometry of phospho-ser880 with GFP: 0.25±0.09 (arbitrary units); with Plk2: 0.89±0.16; p=0.008. (g) COS-7 cells were transfected as shown at top, and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (sGluA2). (h) Quantification of data in (g); surface GluA2 was normalized to total GluA2, then plotted as percent of control. (i) HEK cells were transfected as indicated across top row. Cells were immunostained for sGluA2 (top row, red) and total GluA2 (tGluA2, middle row, green); overlay is shown in yellow (bottom row). Scale bar, 10 μm. (j) Quantification of surface to total GluA2 ratio from (i). **p<0.01; ***p<0.001.
Anti Myc Tag, supplied by Valiant Co Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit  (Valiant Co Ltd)
94
Valiant Co Ltd rabbit
(a, b) Lysates from COS-7 cells transfected as shown at top were analyzed by IP and IB for proteins as indicated. For (b), densitometry of Plk2 associated <t>with</t> <t>NSF</t> plus GluA2: 3.45±0.34 (arbitrary units); minus GluA2: 1.57±0.15; p=0.03. (c) Lysates from cultured hippocampal neurons, infected for 18–20 h with Sindbis virus expressing Plk2 or <t>GFP,</t> were subjected to IP and IB as indicated. (d) Cultured hippocampal neurons were treated with PTX or vehicle (Veh.) for 24 h, and cell lysates used for IP and IB as indicated. (e) Lysates from cultured hippocampal neurons, infected for 18-20 h with Sind-Plk2 or -GFP, were subjected to IP and IB as indicated. Full-length blots are presented in . All MW in kDa. IN, Input, 5% lysate used for IPs. (f) Lysates of cultured hippocampal neurons infected with Sind-GFP or -Plk2 for 18–20 h were immunoblotted as indicated. Densitometry of phospho-ser880 with GFP: 0.25±0.09 (arbitrary units); with Plk2: 0.89±0.16; p=0.008. (g) COS-7 cells were transfected as shown at top, and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (sGluA2). (h) Quantification of data in (g); surface GluA2 was normalized to total GluA2, then plotted as percent of control. (i) HEK cells were transfected as indicated across top row. Cells were immunostained for sGluA2 (top row, red) and total GluA2 (tGluA2, middle row, green); overlay is shown in yellow (bottom row). Scale bar, 10 μm. (j) Quantification of surface to total GluA2 ratio from (i). **p<0.01; ***p<0.001.
Rabbit, supplied by Valiant Co Ltd, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti his tag  (Valiant Co Ltd)
94
Valiant Co Ltd anti his tag
(a, b) Lysates from COS-7 cells transfected as shown at top were analyzed by IP and IB for proteins as indicated. For (b), densitometry of Plk2 associated <t>with</t> <t>NSF</t> plus GluA2: 3.45±0.34 (arbitrary units); minus GluA2: 1.57±0.15; p=0.03. (c) Lysates from cultured hippocampal neurons, infected for 18–20 h with Sindbis virus expressing Plk2 or <t>GFP,</t> were subjected to IP and IB as indicated. (d) Cultured hippocampal neurons were treated with PTX or vehicle (Veh.) for 24 h, and cell lysates used for IP and IB as indicated. (e) Lysates from cultured hippocampal neurons, infected for 18-20 h with Sind-Plk2 or -GFP, were subjected to IP and IB as indicated. Full-length blots are presented in . All MW in kDa. IN, Input, 5% lysate used for IPs. (f) Lysates of cultured hippocampal neurons infected with Sind-GFP or -Plk2 for 18–20 h were immunoblotted as indicated. Densitometry of phospho-ser880 with GFP: 0.25±0.09 (arbitrary units); with Plk2: 0.89±0.16; p=0.008. (g) COS-7 cells were transfected as shown at top, and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (sGluA2). (h) Quantification of data in (g); surface GluA2 was normalized to total GluA2, then plotted as percent of control. (i) HEK cells were transfected as indicated across top row. Cells were immunostained for sGluA2 (top row, red) and total GluA2 (tGluA2, middle row, green); overlay is shown in yellow (bottom row). Scale bar, 10 μm. (j) Quantification of surface to total GluA2 ratio from (i). **p<0.01; ***p<0.001.
Anti His Tag, supplied by Valiant Co Ltd, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti-ddddk tag (flag tag) mab  (Valiant Co Ltd)
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MP Biomedicals DDDDK-Tag (FLAG Tag) Rabbit mAb offers high specificity and potency and consistency against FLAG Tag, which has been experimentally validated in Western blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), and Immunofluorescence (IF) assays.
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human apo a1  (Valiant Co Ltd)
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Human APOA1/Apolipoprotein A1 protein participates in the reverse transport of cholesterol from tissues to the liver for excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for the lecithin cholesterol acyltransferase.
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rabbit anti-gst tag mab  (Valiant Co Ltd)
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MP Biomedicals GST-Tag Rabbit monoclonal antibody offers high specificity and potency and consistency against the GST tag, which has been experimentally validated in Western blotting (WB) and Immunoprecipitation (IP) assays.
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mouse anti mcherry-tag mab  (Valiant Co Ltd)
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Image Search Results


(a, b) Lysates from COS-7 cells transfected as shown at top were analyzed by IP and IB for proteins as indicated. For (b), densitometry of Plk2 associated with NSF plus GluA2: 3.45±0.34 (arbitrary units); minus GluA2: 1.57±0.15; p=0.03. (c) Lysates from cultured hippocampal neurons, infected for 18–20 h with Sindbis virus expressing Plk2 or GFP, were subjected to IP and IB as indicated. (d) Cultured hippocampal neurons were treated with PTX or vehicle (Veh.) for 24 h, and cell lysates used for IP and IB as indicated. (e) Lysates from cultured hippocampal neurons, infected for 18-20 h with Sind-Plk2 or -GFP, were subjected to IP and IB as indicated. Full-length blots are presented in . All MW in kDa. IN, Input, 5% lysate used for IPs. (f) Lysates of cultured hippocampal neurons infected with Sind-GFP or -Plk2 for 18–20 h were immunoblotted as indicated. Densitometry of phospho-ser880 with GFP: 0.25±0.09 (arbitrary units); with Plk2: 0.89±0.16; p=0.008. (g) COS-7 cells were transfected as shown at top, and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (sGluA2). (h) Quantification of data in (g); surface GluA2 was normalized to total GluA2, then plotted as percent of control. (i) HEK cells were transfected as indicated across top row. Cells were immunostained for sGluA2 (top row, red) and total GluA2 (tGluA2, middle row, green); overlay is shown in yellow (bottom row). Scale bar, 10 μm. (j) Quantification of surface to total GluA2 ratio from (i). **p<0.01; ***p<0.001.

Journal: Nature neuroscience

Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

doi: 10.1038/nn.2624

Figure Lengend Snippet: (a, b) Lysates from COS-7 cells transfected as shown at top were analyzed by IP and IB for proteins as indicated. For (b), densitometry of Plk2 associated with NSF plus GluA2: 3.45±0.34 (arbitrary units); minus GluA2: 1.57±0.15; p=0.03. (c) Lysates from cultured hippocampal neurons, infected for 18–20 h with Sindbis virus expressing Plk2 or GFP, were subjected to IP and IB as indicated. (d) Cultured hippocampal neurons were treated with PTX or vehicle (Veh.) for 24 h, and cell lysates used for IP and IB as indicated. (e) Lysates from cultured hippocampal neurons, infected for 18-20 h with Sind-Plk2 or -GFP, were subjected to IP and IB as indicated. Full-length blots are presented in . All MW in kDa. IN, Input, 5% lysate used for IPs. (f) Lysates of cultured hippocampal neurons infected with Sind-GFP or -Plk2 for 18–20 h were immunoblotted as indicated. Densitometry of phospho-ser880 with GFP: 0.25±0.09 (arbitrary units); with Plk2: 0.89±0.16; p=0.008. (g) COS-7 cells were transfected as shown at top, and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (sGluA2). (h) Quantification of data in (g); surface GluA2 was normalized to total GluA2, then plotted as percent of control. (i) HEK cells were transfected as indicated across top row. Cells were immunostained for sGluA2 (top row, red) and total GluA2 (tGluA2, middle row, green); overlay is shown in yellow (bottom row). Scale bar, 10 μm. (j) Quantification of surface to total GluA2 ratio from (i). **p<0.01; ***p<0.001.

Article Snippet: The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore).

Techniques: Transfection, Cell Culture, Infection, Expressing, Labeling, Isolation, Avidin-Biotin Assay

(a, b) Hippocampal cultured neurons were infected with Sind-Plk2 (WT or KN) or Sind-GFP, as indicated at top. Immunostaining is shown for exogenous protein (green) and either surface GluA2, surface GluA1, total GluA2, or total GluA1 (violet) as indicated. Colocalization appears white in merged images (upper rows). Higher magnification views of representative dendrites are shown below for infected and nearby uninfected control neurons. Arrows indicate infected neurons. (c, d) Quantification of results from (a, b). Average immunofluorescence intensity for surface and total GluA2 (c) and for GluA1 (d); N = 40–50 neurons per condition. (e) Quantification of surface GluA2 and GluA1 immunofluorescence intensity measured on the cell body for same neurons as above. ***p<0.001, *p<0.05. Scale bars, 20 μm (wide view), 5 μm (magnified images).

Journal: Nature neuroscience

Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

doi: 10.1038/nn.2624

Figure Lengend Snippet: (a, b) Hippocampal cultured neurons were infected with Sind-Plk2 (WT or KN) or Sind-GFP, as indicated at top. Immunostaining is shown for exogenous protein (green) and either surface GluA2, surface GluA1, total GluA2, or total GluA1 (violet) as indicated. Colocalization appears white in merged images (upper rows). Higher magnification views of representative dendrites are shown below for infected and nearby uninfected control neurons. Arrows indicate infected neurons. (c, d) Quantification of results from (a, b). Average immunofluorescence intensity for surface and total GluA2 (c) and for GluA1 (d); N = 40–50 neurons per condition. (e) Quantification of surface GluA2 and GluA1 immunofluorescence intensity measured on the cell body for same neurons as above. ***p<0.001, *p<0.05. Scale bars, 20 μm (wide view), 5 μm (magnified images).

Article Snippet: The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore).

Techniques: Cell Culture, Infection, Immunostaining, Immunofluorescence

(a) Immunolabeling as indicated for endogenous Plk2 and surface or total GluAs in cultured hippocampal neurons under basal conditions and after stimulation with picrotoxin (PTX) for 24 h. Higher magnification views of representative dendrites are shown below. Arrows indicate cell body of neuron shown. (b) Quantification of data in (a). ***p<0.001, *p<0.05; N=25 neurons per condition. (c) Inverse correlation between Plk2 expression and surface GluA2 levels in individual cells under basal and PTX induced conditions. (d) Hippocampal neurons were transfected with pLL3.7 empty vector or Plk2 RNAi as indicated, along with pEGFP to mark transfected cells. Neurons were treated with PTX or vehicle (basal) and immunostained for GFP and sGluA2 as indicated. Arrows indicate transfected neuron cell body. (e) Quantification of data from (d). N=10–15 neurons per condition; ***p<0.001. Scale bars, 10 μm (wide view), 5 μm (magnified images).

Journal: Nature neuroscience

Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

doi: 10.1038/nn.2624

Figure Lengend Snippet: (a) Immunolabeling as indicated for endogenous Plk2 and surface or total GluAs in cultured hippocampal neurons under basal conditions and after stimulation with picrotoxin (PTX) for 24 h. Higher magnification views of representative dendrites are shown below. Arrows indicate cell body of neuron shown. (b) Quantification of data in (a). ***p<0.001, *p<0.05; N=25 neurons per condition. (c) Inverse correlation between Plk2 expression and surface GluA2 levels in individual cells under basal and PTX induced conditions. (d) Hippocampal neurons were transfected with pLL3.7 empty vector or Plk2 RNAi as indicated, along with pEGFP to mark transfected cells. Neurons were treated with PTX or vehicle (basal) and immunostained for GFP and sGluA2 as indicated. Arrows indicate transfected neuron cell body. (e) Quantification of data from (d). N=10–15 neurons per condition; ***p<0.001. Scale bars, 10 μm (wide view), 5 μm (magnified images).

Article Snippet: The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore).

Techniques: Immunolabeling, Cell Culture, Expressing, Transfection, Plasmid Preparation

(a) Time-course of GluA2 internalization following 18–20 h expression of Sindbis virus-expressed GFP or Plk2. Immunolabeling of infected neurons (GFP or Plk2) and internalized GluA2 or steady-state sGluA2 are indicated. Bottom, higher magnification views of representative dendrites for Sind-Plk2- or Sind-GFP-infected neurons. Arrows indicate infected neurons. Scale bars, 10 μm (wide view), 5 μm (magnified). (b) GluA2 recycling shown 30 min after acid strip of remaining surface labeling. Immunolabeling of infected cells (GFP or Plk2, top row) and recycled GluA2 (bottom row) are shown. (c) Quantification of (b); control with acid strip and no incubation time to allow for recycling (‘Strip’) demonstrates successful stripping of remaining surface primary antibody. Ratio of recycled receptor to available intracellular receptor is shown by line graph on secondary axis. (d) Surface biotinylation of cultured hippocampal neurons. Sister cultures were infected with Sind-GFP, Sind-Plk2-KN, or Sind-Plk2 for 20 h and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (tGluA2 and sGluA2), total and surface GluA1 (tGluA1 and sGluA1), and Plk2 as indicated. No Plk2 was detected in surface fractions (not shown). Both Plk2-KN and Plk2 significantly reduced sGluA2. (e) Quantification of data from (d), normalized to total receptor expression in GFP control cells. (f) Quantification of (a) plotted as ratio of internalized to available surface GluA2 immunofluorescence; N=18–25 neurons per condition. **p<0.01; ***p<0.001.

Journal: Nature neuroscience

Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

doi: 10.1038/nn.2624

Figure Lengend Snippet: (a) Time-course of GluA2 internalization following 18–20 h expression of Sindbis virus-expressed GFP or Plk2. Immunolabeling of infected neurons (GFP or Plk2) and internalized GluA2 or steady-state sGluA2 are indicated. Bottom, higher magnification views of representative dendrites for Sind-Plk2- or Sind-GFP-infected neurons. Arrows indicate infected neurons. Scale bars, 10 μm (wide view), 5 μm (magnified). (b) GluA2 recycling shown 30 min after acid strip of remaining surface labeling. Immunolabeling of infected cells (GFP or Plk2, top row) and recycled GluA2 (bottom row) are shown. (c) Quantification of (b); control with acid strip and no incubation time to allow for recycling (‘Strip’) demonstrates successful stripping of remaining surface primary antibody. Ratio of recycled receptor to available intracellular receptor is shown by line graph on secondary axis. (d) Surface biotinylation of cultured hippocampal neurons. Sister cultures were infected with Sind-GFP, Sind-Plk2-KN, or Sind-Plk2 for 20 h and cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted for total and surface GluA2 (tGluA2 and sGluA2), total and surface GluA1 (tGluA1 and sGluA1), and Plk2 as indicated. No Plk2 was detected in surface fractions (not shown). Both Plk2-KN and Plk2 significantly reduced sGluA2. (e) Quantification of data from (d), normalized to total receptor expression in GFP control cells. (f) Quantification of (a) plotted as ratio of internalized to available surface GluA2 immunofluorescence; N=18–25 neurons per condition. **p<0.01; ***p<0.001.

Article Snippet: The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore).

Techniques: Expressing, Immunolabeling, Infection, Stripping Membranes, Labeling, Incubation, Cell Culture, Isolation, Avidin-Biotin Assay, Immunofluorescence

(a) Cultured hippocampal neurons were transfected with GFP or Plk2 as indicated and cotransfected with pepRA to block NSF-GluA2 interaction or control peptide pepKA. Cells were immunolabeled as indicated for sGluA2 (violet) and for GFP or Plk2 (green). Colocalization appears white in merged images (upper row). Arrows indicate transfected cells. Higher magnification views of representative dendrites are shown below wide view images. Scale bars, 10 μm (wide view), 5 μm (magnified images). (b) Quantification of data from (a) including data from pep2m expression. N=10–15 neurons per condition; ***p<0.001. (c) Amino acid sequences of peptides used.

Journal: Nature neuroscience

Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

doi: 10.1038/nn.2624

Figure Lengend Snippet: (a) Cultured hippocampal neurons were transfected with GFP or Plk2 as indicated and cotransfected with pepRA to block NSF-GluA2 interaction or control peptide pepKA. Cells were immunolabeled as indicated for sGluA2 (violet) and for GFP or Plk2 (green). Colocalization appears white in merged images (upper row). Arrows indicate transfected cells. Higher magnification views of representative dendrites are shown below wide view images. Scale bars, 10 μm (wide view), 5 μm (magnified images). (b) Quantification of data from (a) including data from pep2m expression. N=10–15 neurons per condition; ***p<0.001. (c) Amino acid sequences of peptides used.

Article Snippet: The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore).

Techniques: Cell Culture, Transfection, Blocking Assay, Immunolabeling, Expressing

(a) Cultured hippocampal neurons transfected with GFP, Plk2-W504F or Plk2c as indicated were immunostained for sGluA2 (bottom panels, violet) and for exogenous proteins (middle panels, green). Colocalization appears white in merged images (upper panels). Magnified views of representative dendrites shown at bottom. Arrows indicate transfected neurons. Scale bars, 10 μm (wide), 5 μm (magnified). (b) Quantification of data from (a); N=15–20 neurons per condition; ***p<0.001. (c) COS-7 cells transfected with NSF and/or Plk2 W504F were used for immunoprecipitation (IP) with NSF antibodies or control IgG and immunoblotted (IB) as indicated. Full-length blots are presented in . (d) Deletion analysis of Plk2c-pBHA using yeast two hybrid (Y2H) assays against pGAD-NSF (i, ii). Strongly positive interactions, colored bars. Interaction strength is reported by X-gal reaction time (+++, <10 min; ++, 10–45 min; +, 45–75 min; +/−, 75–120 min; −, >120 min). Numbering represents amino acid residues. (iii, iv) Site-directed mutagenesis targeting conserved residues between Plk2 and Plk3 (bold red in panel v). (v) Amino acid sequence of the PBind site. (e) Y2H analysis of Plk2 binding site on NSF. PBind in pBHA was tested against pGAD constructs of individual NSF domains (N, D1 and D2 as shown) or point mutations in D1 ATPase domain (K266A or E329A) in full-length NSF.

Journal: Nature neuroscience

Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

doi: 10.1038/nn.2624

Figure Lengend Snippet: (a) Cultured hippocampal neurons transfected with GFP, Plk2-W504F or Plk2c as indicated were immunostained for sGluA2 (bottom panels, violet) and for exogenous proteins (middle panels, green). Colocalization appears white in merged images (upper panels). Magnified views of representative dendrites shown at bottom. Arrows indicate transfected neurons. Scale bars, 10 μm (wide), 5 μm (magnified). (b) Quantification of data from (a); N=15–20 neurons per condition; ***p<0.001. (c) COS-7 cells transfected with NSF and/or Plk2 W504F were used for immunoprecipitation (IP) with NSF antibodies or control IgG and immunoblotted (IB) as indicated. Full-length blots are presented in . (d) Deletion analysis of Plk2c-pBHA using yeast two hybrid (Y2H) assays against pGAD-NSF (i, ii). Strongly positive interactions, colored bars. Interaction strength is reported by X-gal reaction time (+++, <10 min; ++, 10–45 min; +, 45–75 min; +/−, 75–120 min; −, >120 min). Numbering represents amino acid residues. (iii, iv) Site-directed mutagenesis targeting conserved residues between Plk2 and Plk3 (bold red in panel v). (v) Amino acid sequence of the PBind site. (e) Y2H analysis of Plk2 binding site on NSF. PBind in pBHA was tested against pGAD constructs of individual NSF domains (N, D1 and D2 as shown) or point mutations in D1 ATPase domain (K266A or E329A) in full-length NSF.

Article Snippet: The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore).

Techniques: Cell Culture, Transfection, Immunoprecipitation, Mutagenesis, Sequencing, Binding Assay, Construct

(a) Lysates of COS-7 cells expressing NSF and GFP or GFP-PBind were subjected to immunoprecipitation (IP) with GFP antibodies and immunoblotted (IB) as indicated. Input, 5% of lysate for IP. Full-length blots are presented in . (b) Pulldown of brain lysates with MBP or MBP-PBind and analyzed by IB for NSF and by Coomassie stain for MBP fusion proteins. Note MBP contains a 73aa polylinker C-terminal tail and therefore runs larger than MBP-PBind. (c) Cultured hippocampal neurons expressing GFP or GFP-PBind as indicated were immunostained for sGluA2 (bottom, violet) and GFP or Plk2 (middle, green). Arrows indicate transfected neurons. Colocalization appears white in merged images (top). Magnified views of representative dendrites at bottom. Scale bars, 10 μm (wide view), 5 μm (magnified). Surface GluA2 immunofluorescence intensities (in arbitrary units) were 82.8±7.3 for GFP, 18.6±3.3 for GFP-PBind; p=4.7×10 -10 ; N=15–20 neurons. (d) Pep-PBind or scrambled control peptide (pep-scr) were analyzed for direct binding to His 6 -NSF by surface plasmon resonance. Averages of peak values were 89.4±7.6 response units for pep-PBind; -1.3±1.1 for pep-scr (negative value due to slight rundown during the experiment); p=0.0003, N=3). (e) Normalized AMPAR EPSCs peak amplitude vs time (pep-PBind, N=9, point 1 vs. point 2: p=0.0001; pep-scr, N=6, point 1 vs. point 2: p=0.17). (f) Representative EPSCs from individual neurons recorded at indicated times in the peak amplitude vs. time plot in the presence of intracellular pep-PBind (top) or pep-scr (bottom).

Journal: Nature neuroscience

Article Title: Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation

doi: 10.1038/nn.2624

Figure Lengend Snippet: (a) Lysates of COS-7 cells expressing NSF and GFP or GFP-PBind were subjected to immunoprecipitation (IP) with GFP antibodies and immunoblotted (IB) as indicated. Input, 5% of lysate for IP. Full-length blots are presented in . (b) Pulldown of brain lysates with MBP or MBP-PBind and analyzed by IB for NSF and by Coomassie stain for MBP fusion proteins. Note MBP contains a 73aa polylinker C-terminal tail and therefore runs larger than MBP-PBind. (c) Cultured hippocampal neurons expressing GFP or GFP-PBind as indicated were immunostained for sGluA2 (bottom, violet) and GFP or Plk2 (middle, green). Arrows indicate transfected neurons. Colocalization appears white in merged images (top). Magnified views of representative dendrites at bottom. Scale bars, 10 μm (wide view), 5 μm (magnified). Surface GluA2 immunofluorescence intensities (in arbitrary units) were 82.8±7.3 for GFP, 18.6±3.3 for GFP-PBind; p=4.7×10 -10 ; N=15–20 neurons. (d) Pep-PBind or scrambled control peptide (pep-scr) were analyzed for direct binding to His 6 -NSF by surface plasmon resonance. Averages of peak values were 89.4±7.6 response units for pep-PBind; -1.3±1.1 for pep-scr (negative value due to slight rundown during the experiment); p=0.0003, N=3). (e) Normalized AMPAR EPSCs peak amplitude vs time (pep-PBind, N=9, point 1 vs. point 2: p=0.0001; pep-scr, N=6, point 1 vs. point 2: p=0.17). (f) Representative EPSCs from individual neurons recorded at indicated times in the peak amplitude vs. time plot in the presence of intracellular pep-PBind (top) or pep-scr (bottom).

Article Snippet: The following antibodies were purchased commercially: mouse NSF (Calbiochem), mouse GluA2 (BD Pharmingen), mouse and rabbit GFP (Qbiogene and Invitrogen), goat Plk2 (SNK C-18, Santa Cruz Biotechnology), rabbit GluA1 (Calbiochem and Millipore), mouse PSD-95 clone K28/43 (NeuroMab), rabbit GRIP1 (Millipore), mouse PICK1 clone L20/8 (NeuroMab), rabbit phospho-Ser880 GluA2 (Millipore).

Techniques: Expressing, Immunoprecipitation, Staining, Cell Culture, Transfection, Immunofluorescence, Binding Assay, SPR Assay