expression plasmid pcdna3 hl1 Search Results


l1cam gene  (Addgene inc)
93
Addgene inc l1cam gene
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
L1cam Gene, supplied by Addgene inc, 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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Average 93 stars, based on 1 article reviews
l1cam gene - by Bioz Stars, 2026-09
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prl-cmv reference plasmid  (Promega)
90
Promega prl-cmv reference plasmid
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Prl Cmv Reference Plasmid, supplied by Promega, 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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prl-cmv reference plasmid - by Bioz Stars, 2026-09
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pcdna3 1 ms2  (Addgene inc)
96
Addgene inc pcdna3 1 ms2
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Pcdna3 1 Ms2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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scd1 inhibitor a939572  (Biofine International Inc)
90
Biofine International Inc scd1 inhibitor a939572
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Scd1 Inhibitor A939572, supplied by Biofine International Inc, 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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pcmv6 xl5pycard v1  (OriGene)
96
OriGene pcmv6 xl5pycard v1
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Pcmv6 Xl5pycard V1, supplied by OriGene, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pcmv6 xl5pycard v1 - by Bioz Stars, 2026-09
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pcdna3.1 vectors  (Shanghai GenePharma)
90
Shanghai GenePharma pcdna3.1 vectors
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Pcdna3.1 Vectors, supplied by Shanghai GenePharma, 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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pcdna3.1 vectors - by Bioz Stars, 2026-09
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negative control plasmid pcdna3.1 oenc  (GenScript corporation)
90
GenScript corporation negative control plasmid pcdna3.1 oenc
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Negative Control Plasmid Pcdna3.1 Oenc, supplied by GenScript corporation, 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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negative control plasmid pcdna3.1 oenc - by Bioz Stars, 2026-09
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transitw lt1 transfection reagent  (Mirus Bio)
99
Mirus Bio transitw lt1 transfection reagent
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Transitw Lt1 Transfection Reagent, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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recombinant dna reagent pt2 cag  (Addgene inc)
93
Addgene inc recombinant dna reagent pt2 cag
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Recombinant Dna Reagent Pt2 Cag, supplied by Addgene inc, 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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figqy peptides  (GenScript corporation)
90
GenScript corporation figqy peptides
( A ) ELISA binding curve of Aknot-HA to the ectodomain of <t>L1CAM.</t> High-absorbance plates were coated with <t>L1CAM</t> across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).
Figqy Peptides, supplied by GenScript corporation, 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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recombinant protein nap new england peptide inc figqy peptides genscript  (Biosynth Carbosynth)
93
Biosynth Carbosynth recombinant protein nap new england peptide inc figqy peptides genscript
<t>NAP</t> effect on tyrosine phosphorylation (pY) of the 16-amino acid FIQGY-peptide fragment derived from the L1-CD, containing a single tyrosine. (A) 2A2-L1s cells were incubated in the absence and presence of 10−9 M NAP, and extracted cell lysates were incubated with <t>FIGQY-peptide</t> conjugated to agarose beads at room temperature for the indicated time (Methods). FIGQpY-peptide was measured using mouse anti-pY mAb (PY20). The magnitude of pY in FIGQY-peptide was quantified by densitometric analysis of anti-pY antibody-agarose bead bands separated by SDS-PAGE. Densities of FIGQpY-peptide bands were normalized to values obtained at time 0. Shown are mean ± SEM % FIGQpY-peptide compared to values at time 0 from 5 independent experiments (F =9.84, p< 0.0001). Peak phosphorylation occurred at 10 minutes following NAP treatment of intact cells. (B) Dose response curve for NAP phosphorylation of FIQGY-peptide by cell lysate from NAP-treated 2A2-L1s cells. Reactions were carried out for 10 min at room temperature in the presence of the indicated concentrations of NAP. pY levels were normalized to values in cells that were not treated with NAP (lane 0). Shown is a representative gel from 12 experiments. Densitometry was obtained from 7 – 12 independent experiments (F = 2.48, p < 0.05). (C) Lysate of cerebral cortex from post-natal day 10 rat pups was incubated in the absence and presence of 10−12 M NAP for 10 minutes. NAP treatment of rat brain lysates significantly increased phosphorylation of FIGQY-peptide (n=7; **t=2.888, ** p < 0.01). (D) 2A2-L1s cells were incubated for 15 minutes at room temperature in the absence and presence of 10−9 M NAP and 20 uM PP2, and cell lysates were then incubated with FIGQY-peptide to determine the effect of drug treatments on pY; *t=3.45, *p=0.010, n=8; **t=3.55, **p=0.0085, n=8.
Recombinant Protein Nap New England Peptide Inc Figqy Peptides Genscript, supplied by Biosynth Carbosynth, 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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Image Search Results


( A ) ELISA binding curve of Aknot-HA to the ectodomain of L1CAM. High-absorbance plates were coated with L1CAM across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).

Journal: Science Advances

Article Title: Vibrio MARTX toxin binding of biantennary N-glycans at host cell surfaces

doi: 10.1126/sciadv.adt0063

Figure Lengend Snippet: ( A ) ELISA binding curve of Aknot-HA to the ectodomain of L1CAM. High-absorbance plates were coated with L1CAM across a concentration gradient, reaching up to 1 μg/ml. The binding intensities Aknot-HA were assessed across a range of specified concentrations. Data points are presented as means ± SD from n = 3 individual experiments and fitted to a one-site specific binding least-squares fit model in Prism 10. ( B ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA; surface expression of L1CAM was detected by phycoerythrin (PE)–conjugated anti-L1CAM. ( C ) Immunofluorescence imaging of Aknot-HA and L1CAM on nonpermeabilized HEK293T cells transfected with an empty vector (pcDNA3.1) or the vector with L1CAM coding sequence. After 48 hours, cells were incubated with 500 nM Aknot-HA. ( D ) Correlation of L1CAM and Aknot-HA mean pixel intensity for each cell in (C) quantified from n = 3 individual experiments analyzed by CellProfiler. Simple linear regression was fitted in Prism 10, best fit was shown in solid line, and 95% confidence intervals were shown in dashed line ( R 2 = 0.85). ( E ) Quantification of flow cytometry analysis of Aknot-HA binding to control and L1CAM −/− HeLa Cas9 cells detected by Alexa Fluor 647–conjugated anti-HA antibody. The average MFI was normalized to 25 nM Aknot-HA binding to control cells for each experiment. Data are presented as means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t tests).

Article Snippet: For transfection, 2.5 μg of plasmid DNA containing the L1CAM gene (Addgene, catalog no. 89411), CDH1-GFP (Addgene, catalog no. 28009), or the corresponding empty vector control pcDNA3 was prepared.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Flow Cytometry, Transfection, Plasmid Preparation, Sequencing, Incubation, Expressing, Immunofluorescence, Imaging, Control

( A ) ELISA binding curve of Aknot-HA to the ectodomain of L1CAM treated with and without PNGaseF. Plates were coated with L1CAM across a concentration gradient. The binding intensities of Aknot-HA were assessed at 1 μM. Means ± SD from n = 3 individual experiments fitted to a one-site specific binding least-squares fit model. The N-glycosylation sites on the L1CAM ectodomain are indicated as red straight lines, with the yellow box indicating the cell membrane. ( B ) Flow cytometry analysis of 100 nM Aknot-HA binding to HEK293T and GNT1 −/− cells. Means ± SD from n = 3 individual experiments (*** P < 0.001, Student’s t test). ( C ) Flow cytometry analysis of Aknot-HA binding to HEK293T and GNT1 −/− cells. Both cell lines were transfected with either an empty vector (pcDNA3.1) or the vector encoding the L1CAM sequence. Forty-eight hours after transfections, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA. Means ± SD from n = 3 individual experiments (*** P < 0.001, Student’s t test). ( D ) Flow cytometry analysis of 100 nM Aknot-HA binding to L1CAM −/− HeLa Cas9 cells treated with or without kifunensine at 37°C overnight. Means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t test). ( E ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty pcDNA3.1 vector (Vehicle) or the vector carrying the gene fusion CDH1 - GFP , which encodes green fluorescent protein (GFP)–tagged E-cadherin (E-CAD). Forty-eight hours after transfections, cells were incubated with 100 nM Aknot-HA. The binding signal of Aknot-HA was detected by the APC channel. The ectopic expression of GFP-tagged E-cadherin was detected by the fluorescein isothiocyanate channel. The cartoon on the left illustrates the N-glycosylation sites (red straight lines) on the E-cadherin ectodomain, with the yellow box indicating the cell membrane.

Journal: Science Advances

Article Title: Vibrio MARTX toxin binding of biantennary N-glycans at host cell surfaces

doi: 10.1126/sciadv.adt0063

Figure Lengend Snippet: ( A ) ELISA binding curve of Aknot-HA to the ectodomain of L1CAM treated with and without PNGaseF. Plates were coated with L1CAM across a concentration gradient. The binding intensities of Aknot-HA were assessed at 1 μM. Means ± SD from n = 3 individual experiments fitted to a one-site specific binding least-squares fit model. The N-glycosylation sites on the L1CAM ectodomain are indicated as red straight lines, with the yellow box indicating the cell membrane. ( B ) Flow cytometry analysis of 100 nM Aknot-HA binding to HEK293T and GNT1 −/− cells. Means ± SD from n = 3 individual experiments (*** P < 0.001, Student’s t test). ( C ) Flow cytometry analysis of Aknot-HA binding to HEK293T and GNT1 −/− cells. Both cell lines were transfected with either an empty vector (pcDNA3.1) or the vector encoding the L1CAM sequence. Forty-eight hours after transfections, cells were incubated with 100 nM Aknot-HA or PBS. The binding signal of Aknot-HA was detected by Alexa Fluor 647–conjugated anti-HA. Means ± SD from n = 3 individual experiments (*** P < 0.001, Student’s t test). ( D ) Flow cytometry analysis of 100 nM Aknot-HA binding to L1CAM −/− HeLa Cas9 cells treated with or without kifunensine at 37°C overnight. Means ± SD from n = 3 individual experiments (**** P < 0.0001, Student’s t test). ( E ) Representative flow cytometry plots of Aknot-HA binding to HEK293T cells. Cells were transfected with either an empty pcDNA3.1 vector (Vehicle) or the vector carrying the gene fusion CDH1 - GFP , which encodes green fluorescent protein (GFP)–tagged E-cadherin (E-CAD). Forty-eight hours after transfections, cells were incubated with 100 nM Aknot-HA. The binding signal of Aknot-HA was detected by the APC channel. The ectopic expression of GFP-tagged E-cadherin was detected by the fluorescein isothiocyanate channel. The cartoon on the left illustrates the N-glycosylation sites (red straight lines) on the E-cadherin ectodomain, with the yellow box indicating the cell membrane.

Article Snippet: For transfection, 2.5 μg of plasmid DNA containing the L1CAM gene (Addgene, catalog no. 89411), CDH1-GFP (Addgene, catalog no. 28009), or the corresponding empty vector control pcDNA3 was prepared.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Glycoproteomics, Membrane, Flow Cytometry, Transfection, Plasmid Preparation, Sequencing, Incubation, Expressing

NAP effect on tyrosine phosphorylation (pY) of the 16-amino acid FIQGY-peptide fragment derived from the L1-CD, containing a single tyrosine. (A) 2A2-L1s cells were incubated in the absence and presence of 10−9 M NAP, and extracted cell lysates were incubated with FIGQY-peptide conjugated to agarose beads at room temperature for the indicated time (Methods). FIGQpY-peptide was measured using mouse anti-pY mAb (PY20). The magnitude of pY in FIGQY-peptide was quantified by densitometric analysis of anti-pY antibody-agarose bead bands separated by SDS-PAGE. Densities of FIGQpY-peptide bands were normalized to values obtained at time 0. Shown are mean ± SEM % FIGQpY-peptide compared to values at time 0 from 5 independent experiments (F =9.84, p< 0.0001). Peak phosphorylation occurred at 10 minutes following NAP treatment of intact cells. (B) Dose response curve for NAP phosphorylation of FIQGY-peptide by cell lysate from NAP-treated 2A2-L1s cells. Reactions were carried out for 10 min at room temperature in the presence of the indicated concentrations of NAP. pY levels were normalized to values in cells that were not treated with NAP (lane 0). Shown is a representative gel from 12 experiments. Densitometry was obtained from 7 – 12 independent experiments (F = 2.48, p < 0.05). (C) Lysate of cerebral cortex from post-natal day 10 rat pups was incubated in the absence and presence of 10−12 M NAP for 10 minutes. NAP treatment of rat brain lysates significantly increased phosphorylation of FIGQY-peptide (n=7; **t=2.888, ** p < 0.01). (D) 2A2-L1s cells were incubated for 15 minutes at room temperature in the absence and presence of 10−9 M NAP and 20 uM PP2, and cell lysates were then incubated with FIGQY-peptide to determine the effect of drug treatments on pY; *t=3.45, *p=0.010, n=8; **t=3.55, **p=0.0085, n=8.

Journal: Biological psychiatry

Article Title: Neuroprotective Peptide NAPVSIPQ Antagonizes Ethanol Inhibition of L1 Adhesion by Promoting the Dissociation of L1 and Ankyrin-G

doi: 10.1016/j.biopsych.2019.08.020

Figure Lengend Snippet: NAP effect on tyrosine phosphorylation (pY) of the 16-amino acid FIQGY-peptide fragment derived from the L1-CD, containing a single tyrosine. (A) 2A2-L1s cells were incubated in the absence and presence of 10−9 M NAP, and extracted cell lysates were incubated with FIGQY-peptide conjugated to agarose beads at room temperature for the indicated time (Methods). FIGQpY-peptide was measured using mouse anti-pY mAb (PY20). The magnitude of pY in FIGQY-peptide was quantified by densitometric analysis of anti-pY antibody-agarose bead bands separated by SDS-PAGE. Densities of FIGQpY-peptide bands were normalized to values obtained at time 0. Shown are mean ± SEM % FIGQpY-peptide compared to values at time 0 from 5 independent experiments (F =9.84, p< 0.0001). Peak phosphorylation occurred at 10 minutes following NAP treatment of intact cells. (B) Dose response curve for NAP phosphorylation of FIQGY-peptide by cell lysate from NAP-treated 2A2-L1s cells. Reactions were carried out for 10 min at room temperature in the presence of the indicated concentrations of NAP. pY levels were normalized to values in cells that were not treated with NAP (lane 0). Shown is a representative gel from 12 experiments. Densitometry was obtained from 7 – 12 independent experiments (F = 2.48, p < 0.05). (C) Lysate of cerebral cortex from post-natal day 10 rat pups was incubated in the absence and presence of 10−12 M NAP for 10 minutes. NAP treatment of rat brain lysates significantly increased phosphorylation of FIGQY-peptide (n=7; **t=2.888, ** p < 0.01). (D) 2A2-L1s cells were incubated for 15 minutes at room temperature in the absence and presence of 10−9 M NAP and 20 uM PP2, and cell lysates were then incubated with FIGQY-peptide to determine the effect of drug treatments on pY; *t=3.45, *p=0.010, n=8; **t=3.55, **p=0.0085, n=8.

Article Snippet: Antibody goat polyclonal antibody (pAb) against the L1-CD Santa Cruz Biotech SC-1508, RRID: AB_631086 mAb against the L1-ECD Santa Cruz Biotech SC-53386 RRID: AB_628937 total L1; rabbit pAb against C-terminus of ankyrin-G Santa Cruz Biotech SC-28561, RRID: AB_633909 mAb against spectrin Santa Cruz Biotech SC-46696, RRID: AB_671135 goat pAb against actin Santa Cruz Biotech SC-1616 , RRID: AB630836 mAB against EphB2 Santa Cruz Biotech SC-130752, RRID: AB_2099957 goat pAb to actin Santa Cruz Biotech SC-1616, RRID AB630836 rabbit pAb against Src Santa Cruz Biotech SC-18, RRID:AB_631324 Antibody mAB anti-Phosphotyrosine Abcam ab10321, RRID: AB_297058 HRP-conjugated secondary antibodies against mouse Jackson ImmunoResearch Laboratories 115-035-062, RRID: AB_2338504 rHRP-conjugated secondary antibodies against rabbit Jackson ImmunoResearch Laboratories 711-035-152, RRID: AB_10015282 rHRP-conjugated secondary antibodies against Goat Jackson ImmunoResearch Laboratories 705-035-003, RRID: AB_2340390 mAb 5G3 Maine Biotech Services ascites production service Bacterial or Viral Strain goat anti-mouse IgG conjugated with Alexa Fluor-546 Thermo Fisher Scientific A-11003, RRID: AB_2534071 goat anti-rabbit IgG conjugated with Alexa Fluor 488 Thermo Fisher Scientific R37120 , RRID: AB_2556548 mAB to b-tubulin Cell Signaling Technology #86298, RRID:AB_2715541 Biological Sample Recombinant Human EphB2 Protein Thermo Fisher Scientific PV3625 Kinase assay buffer Thermo Fisher Scientific PV3189 Biological Sample Sprague Dawley rat pups Charles River Breeding, Worcester, MA Cell Line NIH/3T3 American Type Culture Collection (ATCC) CRL-1658 Chemical Compound or Drug Commercial Assay Or Kit Halt Protease and Phosphatase Inhibitor Cocktail Thermo Scientific Cat #: 1862495 Lipofectamine 2000 Invitrogen Cat#: 11668027 NHS-activated dry agarose beasds Thermofisher Cat #: 26196, Deposited Data; Public Database Genetic Reagent Organism/Strain Peptide, Recombinant Protein NAP New England Peptide Inc FIGQY peptides Genscript USA Inc. Recombinant DNA plasmid, pcDNA3+ wild-type human L1 (hL1) cDNA gene Dr. Patricia Maness, The University of Nother Carolina at Chapell Hill LK, Thelen K, Maness PF.

Techniques: Derivative Assay, Incubation, SDS Page

NAP activation of EphB2 phosphorylation of L1. (A) Effect of NAP on association of L1 with EphB2. L1 was immunoprecipitated with mAb 5G3 from 2A2-L1s cells in the absence and presence of 10−9 M NAP, and coimmunoprecipitated proteins were separated and blotted with antibodies to L1 and EphB2. Densities of EphB2 bands were normalized to those for L1, and values for NAP treatment were expressed as a percentage of control values. Shown is the mean ± SEM % increase in L1 association with EphB2 following NAP treatment derived from 8-9 independent experiments; *t=3.17,*p <0.05, n = 8. (B) Dose-dependent stimulation by NAP of tyrosine phosphorylation of FIGQY-peptide by recombinant EphB2 (F = 2.45, p<0.05). pY levels following NAP treatment were normalized to control values (0 NAP). Shown is the mean ± SEM % increase in pY levels following treatment with the indicated concentrations of NAP derived from 6-9 independent experiments (C) Stimulation of EphB2 phosphorylation of L1 by 10−9 M NAP and P7A-NAP (P7A), but not by 10−9 M I6A-NAP (I6A), SAL, or octanol (Oct). Shown is a representative gel and densitometric analysis from 7 independent experiments. pY levels for each drug treatment were normalized to values obtained in the absence of drugs (Control) (F = 2.84, p<0.05); **t = 4.01, **p = 0.0070; *t= 3.01, *p=0.0235, n=7.

Journal: Biological psychiatry

Article Title: Neuroprotective Peptide NAPVSIPQ Antagonizes Ethanol Inhibition of L1 Adhesion by Promoting the Dissociation of L1 and Ankyrin-G

doi: 10.1016/j.biopsych.2019.08.020

Figure Lengend Snippet: NAP activation of EphB2 phosphorylation of L1. (A) Effect of NAP on association of L1 with EphB2. L1 was immunoprecipitated with mAb 5G3 from 2A2-L1s cells in the absence and presence of 10−9 M NAP, and coimmunoprecipitated proteins were separated and blotted with antibodies to L1 and EphB2. Densities of EphB2 bands were normalized to those for L1, and values for NAP treatment were expressed as a percentage of control values. Shown is the mean ± SEM % increase in L1 association with EphB2 following NAP treatment derived from 8-9 independent experiments; *t=3.17,*p <0.05, n = 8. (B) Dose-dependent stimulation by NAP of tyrosine phosphorylation of FIGQY-peptide by recombinant EphB2 (F = 2.45, p<0.05). pY levels following NAP treatment were normalized to control values (0 NAP). Shown is the mean ± SEM % increase in pY levels following treatment with the indicated concentrations of NAP derived from 6-9 independent experiments (C) Stimulation of EphB2 phosphorylation of L1 by 10−9 M NAP and P7A-NAP (P7A), but not by 10−9 M I6A-NAP (I6A), SAL, or octanol (Oct). Shown is a representative gel and densitometric analysis from 7 independent experiments. pY levels for each drug treatment were normalized to values obtained in the absence of drugs (Control) (F = 2.84, p<0.05); **t = 4.01, **p = 0.0070; *t= 3.01, *p=0.0235, n=7.

Article Snippet: Antibody goat polyclonal antibody (pAb) against the L1-CD Santa Cruz Biotech SC-1508, RRID: AB_631086 mAb against the L1-ECD Santa Cruz Biotech SC-53386 RRID: AB_628937 total L1; rabbit pAb against C-terminus of ankyrin-G Santa Cruz Biotech SC-28561, RRID: AB_633909 mAb against spectrin Santa Cruz Biotech SC-46696, RRID: AB_671135 goat pAb against actin Santa Cruz Biotech SC-1616 , RRID: AB630836 mAB against EphB2 Santa Cruz Biotech SC-130752, RRID: AB_2099957 goat pAb to actin Santa Cruz Biotech SC-1616, RRID AB630836 rabbit pAb against Src Santa Cruz Biotech SC-18, RRID:AB_631324 Antibody mAB anti-Phosphotyrosine Abcam ab10321, RRID: AB_297058 HRP-conjugated secondary antibodies against mouse Jackson ImmunoResearch Laboratories 115-035-062, RRID: AB_2338504 rHRP-conjugated secondary antibodies against rabbit Jackson ImmunoResearch Laboratories 711-035-152, RRID: AB_10015282 rHRP-conjugated secondary antibodies against Goat Jackson ImmunoResearch Laboratories 705-035-003, RRID: AB_2340390 mAb 5G3 Maine Biotech Services ascites production service Bacterial or Viral Strain goat anti-mouse IgG conjugated with Alexa Fluor-546 Thermo Fisher Scientific A-11003, RRID: AB_2534071 goat anti-rabbit IgG conjugated with Alexa Fluor 488 Thermo Fisher Scientific R37120 , RRID: AB_2556548 mAB to b-tubulin Cell Signaling Technology #86298, RRID:AB_2715541 Biological Sample Recombinant Human EphB2 Protein Thermo Fisher Scientific PV3625 Kinase assay buffer Thermo Fisher Scientific PV3189 Biological Sample Sprague Dawley rat pups Charles River Breeding, Worcester, MA Cell Line NIH/3T3 American Type Culture Collection (ATCC) CRL-1658 Chemical Compound or Drug Commercial Assay Or Kit Halt Protease and Phosphatase Inhibitor Cocktail Thermo Scientific Cat #: 1862495 Lipofectamine 2000 Invitrogen Cat#: 11668027 NHS-activated dry agarose beasds Thermofisher Cat #: 26196, Deposited Data; Public Database Genetic Reagent Organism/Strain Peptide, Recombinant Protein NAP New England Peptide Inc FIGQY peptides Genscript USA Inc. Recombinant DNA plasmid, pcDNA3+ wild-type human L1 (hL1) cDNA gene Dr. Patricia Maness, The University of Nother Carolina at Chapell Hill LK, Thelen K, Maness PF.

Techniques: Activation Assay, Immunoprecipitation, Derivative Assay, Recombinant

Effect of EphB2 knockdown on NAP antagonism of ethanol inhibition of L1 adhesion. (A) 2A2-L1s cells were treated with an EphB2 siRNA or a scrambled siRNA. L1 and actin were used as loading controls, and densities of protein bands were normalized to those obtained in untreated cells (Control). Shown is a representative gel and mean ± SEM % changes in EphB2 expression derived from 9 independent experiments (F=9.33, p< 0.001); * t =1.07, p <0.0.0044, n=10. (B) EphB2 siRNA specifically reduced while scrambled siRNA had no effect on the phosphorylation of FIGQY-peptide from lysates of 2A2-L1s cells treated with 10−12 M NAP. Values for pY in NAP-treated siRNA-treated cells were normalized to values in control cells that were not treated with NAP (F = 2.77, p < 0.05); *t = 2.47, p =0.04, n=8; t = 2.54, p=0.039, n =8. (C) EphB2-siRNA specifically reduced while scrambled siRNA had no effect on NAP antagonism of ethanol inhibition of L1 adhesion. Values for ethanol inhibition of L1 adhesion in the presence of NAP were normalized to values obtained in the absence of NAP (38.6 ± 6.9%) (F = 7.17, p < 0.0001); t= 9.30, ***p =0.0000, n = 9; t = 7.41, ***p = 0.0001, n =9).

Journal: Biological psychiatry

Article Title: Neuroprotective Peptide NAPVSIPQ Antagonizes Ethanol Inhibition of L1 Adhesion by Promoting the Dissociation of L1 and Ankyrin-G

doi: 10.1016/j.biopsych.2019.08.020

Figure Lengend Snippet: Effect of EphB2 knockdown on NAP antagonism of ethanol inhibition of L1 adhesion. (A) 2A2-L1s cells were treated with an EphB2 siRNA or a scrambled siRNA. L1 and actin were used as loading controls, and densities of protein bands were normalized to those obtained in untreated cells (Control). Shown is a representative gel and mean ± SEM % changes in EphB2 expression derived from 9 independent experiments (F=9.33, p< 0.001); * t =1.07, p <0.0.0044, n=10. (B) EphB2 siRNA specifically reduced while scrambled siRNA had no effect on the phosphorylation of FIGQY-peptide from lysates of 2A2-L1s cells treated with 10−12 M NAP. Values for pY in NAP-treated siRNA-treated cells were normalized to values in control cells that were not treated with NAP (F = 2.77, p < 0.05); *t = 2.47, p =0.04, n=8; t = 2.54, p=0.039, n =8. (C) EphB2-siRNA specifically reduced while scrambled siRNA had no effect on NAP antagonism of ethanol inhibition of L1 adhesion. Values for ethanol inhibition of L1 adhesion in the presence of NAP were normalized to values obtained in the absence of NAP (38.6 ± 6.9%) (F = 7.17, p < 0.0001); t= 9.30, ***p =0.0000, n = 9; t = 7.41, ***p = 0.0001, n =9).

Article Snippet: Antibody goat polyclonal antibody (pAb) against the L1-CD Santa Cruz Biotech SC-1508, RRID: AB_631086 mAb against the L1-ECD Santa Cruz Biotech SC-53386 RRID: AB_628937 total L1; rabbit pAb against C-terminus of ankyrin-G Santa Cruz Biotech SC-28561, RRID: AB_633909 mAb against spectrin Santa Cruz Biotech SC-46696, RRID: AB_671135 goat pAb against actin Santa Cruz Biotech SC-1616 , RRID: AB630836 mAB against EphB2 Santa Cruz Biotech SC-130752, RRID: AB_2099957 goat pAb to actin Santa Cruz Biotech SC-1616, RRID AB630836 rabbit pAb against Src Santa Cruz Biotech SC-18, RRID:AB_631324 Antibody mAB anti-Phosphotyrosine Abcam ab10321, RRID: AB_297058 HRP-conjugated secondary antibodies against mouse Jackson ImmunoResearch Laboratories 115-035-062, RRID: AB_2338504 rHRP-conjugated secondary antibodies against rabbit Jackson ImmunoResearch Laboratories 711-035-152, RRID: AB_10015282 rHRP-conjugated secondary antibodies against Goat Jackson ImmunoResearch Laboratories 705-035-003, RRID: AB_2340390 mAb 5G3 Maine Biotech Services ascites production service Bacterial or Viral Strain goat anti-mouse IgG conjugated with Alexa Fluor-546 Thermo Fisher Scientific A-11003, RRID: AB_2534071 goat anti-rabbit IgG conjugated with Alexa Fluor 488 Thermo Fisher Scientific R37120 , RRID: AB_2556548 mAB to b-tubulin Cell Signaling Technology #86298, RRID:AB_2715541 Biological Sample Recombinant Human EphB2 Protein Thermo Fisher Scientific PV3625 Kinase assay buffer Thermo Fisher Scientific PV3189 Biological Sample Sprague Dawley rat pups Charles River Breeding, Worcester, MA Cell Line NIH/3T3 American Type Culture Collection (ATCC) CRL-1658 Chemical Compound or Drug Commercial Assay Or Kit Halt Protease and Phosphatase Inhibitor Cocktail Thermo Scientific Cat #: 1862495 Lipofectamine 2000 Invitrogen Cat#: 11668027 NHS-activated dry agarose beasds Thermofisher Cat #: 26196, Deposited Data; Public Database Genetic Reagent Organism/Strain Peptide, Recombinant Protein NAP New England Peptide Inc FIGQY peptides Genscript USA Inc. Recombinant DNA plasmid, pcDNA3+ wild-type human L1 (hL1) cDNA gene Dr. Patricia Maness, The University of Nother Carolina at Chapell Hill LK, Thelen K, Maness PF.

Techniques: Inhibition, Expressing, Derivative Assay

KEY RESOURCES TABLE

Journal: Biological psychiatry

Article Title: Neuroprotective Peptide NAPVSIPQ Antagonizes Ethanol Inhibition of L1 Adhesion by Promoting the Dissociation of L1 and Ankyrin-G

doi: 10.1016/j.biopsych.2019.08.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibody goat polyclonal antibody (pAb) against the L1-CD Santa Cruz Biotech SC-1508, RRID: AB_631086 mAb against the L1-ECD Santa Cruz Biotech SC-53386 RRID: AB_628937 total L1; rabbit pAb against C-terminus of ankyrin-G Santa Cruz Biotech SC-28561, RRID: AB_633909 mAb against spectrin Santa Cruz Biotech SC-46696, RRID: AB_671135 goat pAb against actin Santa Cruz Biotech SC-1616 , RRID: AB630836 mAB against EphB2 Santa Cruz Biotech SC-130752, RRID: AB_2099957 goat pAb to actin Santa Cruz Biotech SC-1616, RRID AB630836 rabbit pAb against Src Santa Cruz Biotech SC-18, RRID:AB_631324 Antibody mAB anti-Phosphotyrosine Abcam ab10321, RRID: AB_297058 HRP-conjugated secondary antibodies against mouse Jackson ImmunoResearch Laboratories 115-035-062, RRID: AB_2338504 rHRP-conjugated secondary antibodies against rabbit Jackson ImmunoResearch Laboratories 711-035-152, RRID: AB_10015282 rHRP-conjugated secondary antibodies against Goat Jackson ImmunoResearch Laboratories 705-035-003, RRID: AB_2340390 mAb 5G3 Maine Biotech Services ascites production service Bacterial or Viral Strain goat anti-mouse IgG conjugated with Alexa Fluor-546 Thermo Fisher Scientific A-11003, RRID: AB_2534071 goat anti-rabbit IgG conjugated with Alexa Fluor 488 Thermo Fisher Scientific R37120 , RRID: AB_2556548 mAB to b-tubulin Cell Signaling Technology #86298, RRID:AB_2715541 Biological Sample Recombinant Human EphB2 Protein Thermo Fisher Scientific PV3625 Kinase assay buffer Thermo Fisher Scientific PV3189 Biological Sample Sprague Dawley rat pups Charles River Breeding, Worcester, MA Cell Line NIH/3T3 American Type Culture Collection (ATCC) CRL-1658 Chemical Compound or Drug Commercial Assay Or Kit Halt Protease and Phosphatase Inhibitor Cocktail Thermo Scientific Cat #: 1862495 Lipofectamine 2000 Invitrogen Cat#: 11668027 NHS-activated dry agarose beasds Thermofisher Cat #: 26196, Deposited Data; Public Database Genetic Reagent Organism/Strain Peptide, Recombinant Protein NAP New England Peptide Inc FIGQY peptides Genscript USA Inc. Recombinant DNA plasmid, pcDNA3+ wild-type human L1 (hL1) cDNA gene Dr. Patricia Maness, The University of Nother Carolina at Chapell Hill LK, Thelen K, Maness PF.

Techniques: Recombinant, Protein Kinase Assay, Kinase Assay, Plasmid Preparation, Sequencing, Software, Transfection, Construct, Expressing