Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Schematic of the Cas9/sgRNA targeting of ATP7A. The sgRNA-targeting sequence is shown; the PAM sequence is labeled in red. The results of sequencing of ATP7A genomic regions are shown below the WT sequence. (B) Protein levels of ATP7A in undifferentiated WT and ATP7A +/− ; α-tubulin is a loading control. (C) The total Cu levels in undifferentiated WT and ATP7A +/− and ATP7A −/− cells measured using atomic absorption spectroscopy ( n = 3). (D) The XFM images of phosphate (“P”), used as a control, and Cu in undifferentiated WT and ATP7A +/− cells show differences in the intracellular distribution of Cu ( n = 3); nuclei are indicated by orange circles. (E) The quantitative analysis of fluorescence intensity in the perinuclear region containing bright Cu puncta in WT and ATP7A +/− cells ( n = 8, 3 respectively). (F) The bright-field images of differentiated WT and ATP7A +/− cells at day 9 illustrate enlargement of cells with down-regulated ATP7A. (G) Distribution of cell sizes and (H) triglyceride levels for WT ( n = 98; n = 3) and ATP7A +/− adipocytes ( n = 101; n = 3); triglyceride levels were normalized to the protein levels, and these values were compared to WT cells’ values taken as 1; the ratio is plotted. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; Cu, copper; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; WT, wild type; XFM, X-ray fluorescence microscopy.

Article Snippet: Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001).

Techniques: Sequencing, Labeling, Atomic Absorption Spectroscopy, Fluorescence, CRISPR, Microscopy

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Cu limitation with 10 μM TTM for 48 h decreases the SSAO activity ( n = 3). (B) SSAO activity in the epididymal adipose tissue from 13-wk-old male rats fed with Cu-adequate or low-Cu diet for 8 wk ( n = 7). (C) Down-regulation of ATP7A in ATP7A +/− adipocytes causes a decrease in SSAO activity ( n = 4). (D) Immunocytochemistry shows that SSAO transits the ATP7A-containing compartment on its way to the plasma membrane. Top: ATP7A (green) is localized to the TGN, as evidenced by its colocalization with the TGN marker Syn6 (red); Bottom: SSAO (red) is present at the plasma membrane and inside the cells, where it colocalizes with ATP7A (green). Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. Cu, copper; ns, not significant; SSAO, semicarbazide-sensitive amine oxidase; Syn6, syntaxin 6; TGN, trans -Golgi network; TTM, tetrathiomolybdate; WT, wild type.

Article Snippet: Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001).

Techniques: Activity Assay, Immunocytochemistry, Marker

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) The Cas9/sgRNA-targeting sites in the SSAO gene. The sgRNA-targeting sequence is shown, and the PAM sequence is labeled in red. The results of sequencing of both AOC3 alleles in SSAO −/− cells are shown under the WT sequence. (B) The SSAO activity in WT and SSAO −/− adipocytes ( n = 4). (C) Immunostaining of SSAO (red) and ATP7A (green) in the WT and SSAO −/− adipocytes. (D) Bright-field images of differentiated WT and SSAO −/− cells at day 9. (E) The size distribution and (F) triglyceride levels for the WT ( n = 133; n = 3) and SSAO −/− ( n = 139; n = 3) adipocytes (triglyceride levels were normalized to protein levels, and the values for SSAO −/− cells were compared to the WT control; the ratio is plotted). (G) The size distribution and (I) triglyceride levels for SSAO −/− cells in the basal medium ( n = 178), after treatment with the 0.6 μg/ml sSSAO ( n = 150; n = 3), or 1 μg/ml sSSAO ( n = 184; n = 3); triglyceride levels were normalized to protein levels, and the values for sSSAO-treated cells were compared to values at basal conditions; the ratio is plotted. (H) Effect of sSSAO on the size of SSAO −/− cells at day 9 of differentiation. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; SSAO, semicarbazide-sensitive amine oxidase; sSSAO, recombinant soluble SSAO; WT, wild type.

Article Snippet: Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001).

Techniques: Sequencing, Labeling, Activity Assay, Immunostaining, CRISPR, Recombinant

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Schematic of the Cas9/sgRNA targeting of ATP7A. The sgRNA-targeting sequence is shown; the PAM sequence is labeled in red. The results of sequencing of ATP7A genomic regions are shown below the WT sequence. (B) Protein levels of ATP7A in undifferentiated WT and ATP7A +/− ; α-tubulin is a loading control. (C) The total Cu levels in undifferentiated WT and ATP7A +/− and ATP7A −/− cells measured using atomic absorption spectroscopy ( n = 3). (D) The XFM images of phosphate (“P”), used as a control, and Cu in undifferentiated WT and ATP7A +/− cells show differences in the intracellular distribution of Cu ( n = 3); nuclei are indicated by orange circles. (E) The quantitative analysis of fluorescence intensity in the perinuclear region containing bright Cu puncta in WT and ATP7A +/− cells ( n = 8, 3 respectively). (F) The bright-field images of differentiated WT and ATP7A +/− cells at day 9 illustrate enlargement of cells with down-regulated ATP7A. (G) Distribution of cell sizes and (H) triglyceride levels for WT ( n = 98; n = 3) and ATP7A +/− adipocytes ( n = 101; n = 3); triglyceride levels were normalized to the protein levels, and these values were compared to WT cells’ values taken as 1; the ratio is plotted. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; Cu, copper; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; WT, wild type; XFM, X-ray fluorescence microscopy.

Article Snippet: Proteins were then resolved on 8% Laemmli SDS-PAGE and transferred to PVDF membrane at 90 V for 90 min. Primary antibodies used in immunoblotting were rabbit monoclonal anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse Anti-Na+/K+ ATPase α-1 Antibody (Millipore 06–520), and rabbit Anti-ATP7b antibody [EPR6794] (ab124973).

Techniques: Sequencing, Labeling, Atomic Absorption Spectroscopy, Fluorescence, CRISPR, Microscopy

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Cu limitation with 10 μM TTM for 48 h decreases the SSAO activity ( n = 3). (B) SSAO activity in the epididymal adipose tissue from 13-wk-old male rats fed with Cu-adequate or low-Cu diet for 8 wk ( n = 7). (C) Down-regulation of ATP7A in ATP7A +/− adipocytes causes a decrease in SSAO activity ( n = 4). (D) Immunocytochemistry shows that SSAO transits the ATP7A-containing compartment on its way to the plasma membrane. Top: ATP7A (green) is localized to the TGN, as evidenced by its colocalization with the TGN marker Syn6 (red); Bottom: SSAO (red) is present at the plasma membrane and inside the cells, where it colocalizes with ATP7A (green). Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. Cu, copper; ns, not significant; SSAO, semicarbazide-sensitive amine oxidase; Syn6, syntaxin 6; TGN, trans -Golgi network; TTM, tetrathiomolybdate; WT, wild type.

Article Snippet: Proteins were then resolved on 8% Laemmli SDS-PAGE and transferred to PVDF membrane at 90 V for 90 min. Primary antibodies used in immunoblotting were rabbit monoclonal anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse Anti-Na+/K+ ATPase α-1 Antibody (Millipore 06–520), and rabbit Anti-ATP7b antibody [EPR6794] (ab124973).

Techniques: Activity Assay, Immunocytochemistry, Marker

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) The Cas9/sgRNA-targeting sites in the SSAO gene. The sgRNA-targeting sequence is shown, and the PAM sequence is labeled in red. The results of sequencing of both AOC3 alleles in SSAO −/− cells are shown under the WT sequence. (B) The SSAO activity in WT and SSAO −/− adipocytes ( n = 4). (C) Immunostaining of SSAO (red) and ATP7A (green) in the WT and SSAO −/− adipocytes. (D) Bright-field images of differentiated WT and SSAO −/− cells at day 9. (E) The size distribution and (F) triglyceride levels for the WT ( n = 133; n = 3) and SSAO −/− ( n = 139; n = 3) adipocytes (triglyceride levels were normalized to protein levels, and the values for SSAO −/− cells were compared to the WT control; the ratio is plotted). (G) The size distribution and (I) triglyceride levels for SSAO −/− cells in the basal medium ( n = 178), after treatment with the 0.6 μg/ml sSSAO ( n = 150; n = 3), or 1 μg/ml sSSAO ( n = 184; n = 3); triglyceride levels were normalized to protein levels, and the values for sSSAO-treated cells were compared to values at basal conditions; the ratio is plotted. (H) Effect of sSSAO on the size of SSAO −/− cells at day 9 of differentiation. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; SSAO, semicarbazide-sensitive amine oxidase; sSSAO, recombinant soluble SSAO; WT, wild type.

Article Snippet: Proteins were then resolved on 8% Laemmli SDS-PAGE and transferred to PVDF membrane at 90 V for 90 min. Primary antibodies used in immunoblotting were rabbit monoclonal anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse Anti-Na+/K+ ATPase α-1 Antibody (Millipore 06–520), and rabbit Anti-ATP7b antibody [EPR6794] (ab124973).

Techniques: Sequencing, Labeling, Activity Assay, Immunostaining, CRISPR, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

doi: 10.1074/jbc.M116.727248

Figure Lengend Snippet: Deletion of ATP7A in 3T3-L1 preadipocytes is associated with elevation of cellular copper content and increased oxidation of glutathione in mitochondria. A , CRISPR/Cas9 targeting of exon 2 generated deletion in both alleles of genomic ATP7A in 3T3-L1 cells. B , Western blotting analysis of cell lysates shows the presence of ATP7A in the WT 3T3-L1 cells and a loss of ATP7A expression in CRISPR/Cas9-targeted 3T3-L1 cells. α-Tubulin was used as a loading control. C , copper levels in wild-type (3.4 ± 0.49 ng/mg protein) and ATP7A −/− 3T3-L1 (10.32 ± 0.41 ng/mg protein) cells under basal conditions (****, p < 0.0001; n = 3). D , ratiometric (IR 405/488 ) false-colored image of MTS-GRX1-roGFP2 fluorescence in WT and ATP7A −/− 3T3-L1. Cells were imaged at ×40 magnification. E , oxidation of MTS-GRX1-roGFP2 in mitochondria of wild-type (28.08 ± 17.39, n = 20 cells) and ATP7A −/− 3T3-L1 cells (44.90 ± 20.73, n = 28 cells). **, p = 0.0049. Data are mean ± S.E. of three independent experiments.

Article Snippet: 20 μg of homogenate was separated on 7.5% polyacrylamide gels, transferred onto a PVDF membrane, and incubated overnight at 4 °C with rabbit anti-ATP7A CT77 (Hycult Biotech) to detect ATP7A and with mouse anti-tubulin (Sigma), used as a loading control.

Techniques: CRISPR, Generated, Western Blot, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria *

doi: 10.1074/jbc.M116.727248

Figure Lengend Snippet: Deletion of ATP7A in 3T3-L1 preadipocytes is associated with elevation of cellular copper content and increased oxidation of glutathione in mitochondria. A , CRISPR/Cas9 targeting of exon 2 generated deletion in both alleles of genomic ATP7A in 3T3-L1 cells. B , Western blotting analysis of cell lysates shows the presence of ATP7A in the WT 3T3-L1 cells and a loss of ATP7A expression in CRISPR/Cas9-targeted 3T3-L1 cells. α-Tubulin was used as a loading control. C , copper levels in wild-type (3.4 ± 0.49 ng/mg protein) and ATP7A −/− 3T3-L1 (10.32 ± 0.41 ng/mg protein) cells under basal conditions (****, p < 0.0001; n = 3). D , ratiometric (IR 405/488 ) false-colored image of MTS-GRX1-roGFP2 fluorescence in WT and ATP7A −/− 3T3-L1. Cells were imaged at ×40 magnification. E , oxidation of MTS-GRX1-roGFP2 in mitochondria of wild-type (28.08 ± 17.39, n = 20 cells) and ATP7A −/− 3T3-L1 cells (44.90 ± 20.73, n = 28 cells). **, p = 0.0049. Data are mean ± S.E. of three independent experiments.

Article Snippet: 20 μg of homogenate was separated on 7.5% polyacrylamide gels, transferred onto a PVDF membrane, and incubated overnight at 4 °C with rabbit anti-ATP7A CT77 (Hycult Biotech) to detect ATP7A and with mouse anti-tubulin (Sigma), used as a loading control.

Techniques: CRISPR, Generated, Western Blot, Expressing, Fluorescence

Journal: Nature Communications

Article Title: Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway

doi: 10.1038/ncomms10640

Figure Lengend Snippet: ( a ) Immunostaining of PAM (green) in a chicken spinal cord (HH stage 20 embryo). Traverse sections of chick embryos were analysed. Differentiated cells in marginal zones were identified by postmitotic marker isl1/2 (red). Scale bar, 50 μm. ( b ) Intensity profile along the line indicated by the arrow in a shows overlap between PAM and Isl1/2 expression. Medial and lateral borders are represented as M and L, respevtively. ( c ) Schematic of differentiation of SH-SY5Y cells by sequential treatments with retinoic acid (DIF RA) and BDNF (DIF BDNF). ( d ) Differentiation is associated with upregulation of genes involved in copper fluxes to mitochondria and, especially, the secretory pathway. The mRNA levels were determined by ΔΔCt analysis using GAPDH as a reference gene and normalized to non-differentiated cells (red line). Each value is presented as mean±s.d. ( n =3). ( e ) Protein levels for ATP7A and Atox1 increase upon differentiation. Equal amount of protein was loaded to each lane. Neuronal differentiation was verified by upregulation of MAP2. ( f ) Cellular copper content in differentiated SH-SY5Y cells (BDNF) is higher than in non-differentiated (ND) cells. Copper amounts were determined by atomic absorption and normalized to protein amounts. Data from three independent measurements. ( g , h ) ATP7A is localized within TGN and vesicular structures which are distinct from endosomal or lysosomal compartments. Coimmunostaining with TGN was shown as a representative image (see for other images). Nucleus was stained with DAPI (blue). Scale bar, 10 μm. Colocalization of ATP7A and various markers were evaluated using Pearson's product-moment coefficients. Three replicate samples were prepared and analysed. Each value is presented as mean±s.d. ( n =3).

Article Snippet: Primary antibodies used in cell staining were mouse monoclonal anti-FLAG M2 clone (Sigma, F1804), rabbit polyclonal anti-ATP7A (Hycult Biotech, HP8040), mouse monoclonal anti-ATP7A (Santa Cruz, 376467), mouse monoclonal anti-EEA1 (BD Biosciences, 610456), mouse monoclonal anti-Rab11 (BD Biosciences, 610656), mouse monoclonal anti-Lamp1 (developmental studies hybridoma bank, H4A3-s), and sheep monoclonal anti-TGN46 (Gene Tex, GTX74290).

Techniques: Immunostaining, Marker, Expressing, Staining

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Manhattan plots for hepatic copper score in Labrador retrievers. (A) Manhattan plot of hepatic copper score in 235 Labrador retrievers shows a genome-wide association signal at chromosome 22. (B) Manhattan plot of chromosome 22 shows that the signal comprises an LD block comprising the first 10 Mb of the chromosome. The arrow indicates the location of ATP7B . (C) Mapping results for hepatic copper score at the X chromosome in male dogs shows an association signal at position X:60203319-60356690 (CanFam 3.1). The arrows indicate the location of ATP7A . (D) Mapping results for hepatic copper score at the X chromosome in females shows no substantial association. Note that the solid line indicates the boundary for suggestive genome-wide association at a significance level of P =5×10 −5 . The dotted line indicates the boundary used for determination of the crucial regions at P =5×10 −4 .

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques: GWAS, Blocking Assay

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Hepatic histological copper score in relation to ATP7A and ATP7B genotype in male and female Labrador retrievers

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques:

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Protein domains of ATP7A and ATP7B involved in copper toxicosis in Labrador retrievers. Overview of the ATP7A (A) and ATP7B (B) proteins with the N-terminus, metal-binding domains (MBDs), actuator domain (A), nucleotide-binding domain (N), phosphorylation domain (P) and the C-terminus. The green asterisk indicates the position of the mutations. (A) Alignment of the region containing ATP7A T327I (in green) shows a strong conservation of this amino acid position in the human, rat, mouse, cow, cat and horse. The copper-binding site XMXCXXC (boxed), predicted α-helix (red) and β-sheets (blue) are indicated. (B) Alignment of the region containing ATP7B R1453Q (in green) shows a strong conservation of this amino acid position in the human, rat, mouse, cow, cat and horse. The predicted α-helix (red) and β-sheets (blue) are indicated.

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques: Binding Assay

Journal: Disease Models & Mechanisms

Article Title: The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

doi: 10.1242/dmm.020263

Figure Lengend Snippet: Copper accumulation and retention in canine dermal fibroblasts. (A) Copper influx in canine fibroblasts. Dermal fibroblasts derived from ATP7A:p.Ile327 (ATP7A T327I ) dogs showed significantly more uptake of 64 Cu than dermal fibroblasts derived from dogs with ATP7A:p.Thr327 (ATP7A WT ). The overall difference in copper accumulation was 86% (95% confidence interval 25-176%, P =6.1×10 −3 ). The overall increase in 64 Cu was compared to time point 6 h. After 22 h, the estimated increase in copper was 45% (95% confidence interval 27-64%, P <1.0×10 −4 ), and after 30 h this was 72% (95% confidence interval 45-104%, P <1.0×10 −4 ). Dots represent mean values, and standard deviation is represented by the error bars. (B) Copper efflux in canine fibroblasts. Dermal fibroblasts from ATP7A T327I dogs showed significantly more retention of copper than dermal fibroblasts derived from dogs with ATP7A WT . The overall average export rate in ATP7A T327I was significantly lower than in ATP7A WT ( P =0.011). Dots represent mean percentages, and standard deviation is represented by the error bars. (C) Copper influx in human fibroblasts. Dermal fibroblasts from a human with Menkes disease showed considerably more uptake of copper than dermal fibroblasts derived from a human with ATP7A wild type. (D) Copper efflux in human fibroblasts. Dermal fibroblasts from a human with Menkes disease showed considerably more retention of copper than dermal fibroblasts derived from a person with ATP7A wild type.

Article Snippet: Antibodies were obtained from the following sources: anti-ATP7A (Hycult Biotech, Plymouth Meeting, PA, USA), anti-Human Golgin-97 Mouse Monoclonal CDF4 (Molecular Probes, Paisley, UK), anti-GFP and anti-VAP-A from M.A.

Techniques: Derivative Assay, Standard Deviation