phosphor marcks Search Results


anti phospho myristoylated alanine rich c kinase substrate marcks  (Novus Biologicals)
90
Novus Biologicals anti phospho myristoylated alanine rich c kinase substrate marcks
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Anti Phospho Myristoylated Alanine Rich C Kinase Substrate Marcks, supplied by Novus Biologicals, 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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phospho-marcks (s152/s156) antibody  (Bio-Techne corporation)
92
Bio-Techne corporation phospho-marcks (s152/s156) antibody
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Phospho Marcks (S152/S156) Antibody, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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phospho-pyruvate dehydrogenase kinase (pdk) antibody  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc phospho-pyruvate dehydrogenase kinase (pdk) antibody
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Phospho Pyruvate Dehydrogenase Kinase (Pdk) Antibody, supplied by Cell Signaling Technology 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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phospho marcks ser159 163 polyclonal antibody  (Proteintech)
93
Proteintech phospho marcks ser159 163 polyclonal antibody
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Phospho Marcks Ser159 163 Polyclonal Antibody, supplied by Proteintech, 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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anti-phospho-marcks  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc anti-phospho-marcks
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Anti Phospho Marcks, supplied by Cell Signaling Technology 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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anti p marcks  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc anti p marcks
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Anti P Marcks, supplied by Cell Signaling Technology Inc, 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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11992s total pkc cst  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc 11992s total pkc cst
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
11992s Total Pkc Cst, supplied by Cell Signaling Technology Inc, 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-phospho-marcks (ser46) antibody  (GL Biochem)
90
GL Biochem rabbit anti-phospho-marcks (ser46) antibody
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Rabbit Anti Phospho Marcks (Ser46) Antibody, supplied by GL Biochem, 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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anti-marcks  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc anti-marcks
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Anti Marcks, supplied by Cell Signaling Technology 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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rabbit polyclonal anti-phosphorylated marcks (pser 152/156 , anti-phospho-marcks) antibody  (Millipore)
90
Millipore rabbit polyclonal anti-phosphorylated marcks (pser 152/156 , anti-phospho-marcks) antibody
Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate <t>(MARCKS).</t> mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.
Rabbit Polyclonal Anti Phosphorylated Marcks (Pser 152/156 , Anti Phospho Marcks) Antibody, supplied by Millipore, 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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anti human marcks  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc anti human marcks
Figure 1. <t>MARCKS</t> KD impairs dedifferentiated VSMC migration. Human CASMCs were transfected with 100 nM of MARCKS siRNA (KD) or nontargeting, control siRNA (Control). A, At 72 hours post-siRNA treatment, protein expression of MARCKS was decreased by 93%3% compared to cells treated with control (nontargeting) siRNA. B, MARCKS KD significantly attenuated VSMC migration in the wound-healing scratch assay. C, Cell migration was determined by both reduction of the width of the wound. D, The density of cells within the wound. *P<0.05. Scale bars=500 lm. E, Starved CASMCs were stimulated with PDGF (20 ng/mL for 10 minutes), fixed, and stained with anti-cortactin (green) and anti-MARCKS (red) antibodies. White triangles denote the nucleus which stains for neither cortactin nor MARCKS. Lamellipodia, white arrows, formed around the periphery of the cell in the control group, which were seldom detected after MARCKS KD. White arrow heads denote cell nucleus. Scale bars=10 lm; shown are representative images of 3 independent experiments. F, These morphological changes are best appreciated at higher magnification. MARCKS and cortactin colocalized in lamellipodia (white arrows). Scale bar=10 lm. G, Significantly more cells formed lamellipodia in repsonse to PDGF stimulation in the control group compared to the MARCKS KD group. At least 40 cells were counted in each group per experiment. Data are presented as the mean and SE of 3 independent experiments. CASMCs indicates coronary artery smooth muscle cells; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell. *P<0.001.
Anti Human Marcks, supplied by Cell Signaling Technology 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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phospho-marcks (sc-12971-r)  (Santa Cruz Biotechnology)
90
Santa Cruz Biotechnology phospho-marcks (sc-12971-r)
Phosphorylation of <t>MARCKS</t> is increased in the brains of PKCα-M489V mice. Western blot of lysates of whole brain obtained from nine male and female 3-mo-old wild-type mice (lanes 1–4, males; lanes 5–9, females) or nine male and female genome-edited mice containing a homozygous PKCα-M489V mutation (lanes 10–15, males; lanes 16–18, females). Western blots were probed <t>with</t> <t>antibodies</t> specific to a known PKC phosphorylation site on MARCKS (Ser-159/163) or to total PKCα (Top). Bands were quantified using densitometry and the phospho-MARCKS signal was normalized to total MARCKS signal and PKCα signal was normalized to its β-actin loading control. Normalized data from the depicted Western blot were plotted (Bottom) as average normalized intensity ± SEM (*P < 0.05; n.s., not significantly different using a Student’s t test). Males are indicated in green squares and females in black circles.
Phospho Marcks (Sc 12971 R), supplied by Santa Cruz Biotechnology, 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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Image Search Results


Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate (MARCKS). mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.

Journal: Journal of Lipid Research

Article Title: Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

doi: 10.1194/jlr.m600326-jlr200

Figure Lengend Snippet: Fig. 5. HDL, but not AcLDL, stimulates phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C- kinase substrate (MARCKS). mSR-BI-overexpressing (ldlA[mSR- BI]) cells, prepared as described in the legend to Fig. 4, were given serum-free Hams F12 medium containing either no additions (lane 1), 0.5 mM PMA, HDL, or AcLDL (each at 50 mg/ml), 5 mM RO31-8220, a 1:1000 dilution of an anti-SR-BI blocking antiserum, or combinations as indicated. Cells were incubated for 3 min, at which time they were rapidly chilled on ice, then washed, lysed, and analyzed by SDS-PAGE and immunoblotting for either phospho- MARCKS or b-actin. The data are representative of multiple experi- ments. For the experiment shown, lanes 9 and 10 were from a different portion of the same gel.

Article Snippet: Rabbit anti-SR-BI antibody (NB400-101) and anti-phospho-myristoylated alanine-rich C-kinase substrate (MARCKS) (NB500-140) were from Novus Biologicals, Inc. (distributed by Cedarlane Laboratories Ltd, Hornby, Ontario, Canada).

Techniques: Phospho-proteomics, Blocking Assay, Incubation, SDS Page, Western Blot

Figure 1. MARCKS KD impairs dedifferentiated VSMC migration. Human CASMCs were transfected with 100 nM of MARCKS siRNA (KD) or nontargeting, control siRNA (Control). A, At 72 hours post-siRNA treatment, protein expression of MARCKS was decreased by 93%3% compared to cells treated with control (nontargeting) siRNA. B, MARCKS KD significantly attenuated VSMC migration in the wound-healing scratch assay. C, Cell migration was determined by both reduction of the width of the wound. D, The density of cells within the wound. *P<0.05. Scale bars=500 lm. E, Starved CASMCs were stimulated with PDGF (20 ng/mL for 10 minutes), fixed, and stained with anti-cortactin (green) and anti-MARCKS (red) antibodies. White triangles denote the nucleus which stains for neither cortactin nor MARCKS. Lamellipodia, white arrows, formed around the periphery of the cell in the control group, which were seldom detected after MARCKS KD. White arrow heads denote cell nucleus. Scale bars=10 lm; shown are representative images of 3 independent experiments. F, These morphological changes are best appreciated at higher magnification. MARCKS and cortactin colocalized in lamellipodia (white arrows). Scale bar=10 lm. G, Significantly more cells formed lamellipodia in repsonse to PDGF stimulation in the control group compared to the MARCKS KD group. At least 40 cells were counted in each group per experiment. Data are presented as the mean and SE of 3 independent experiments. CASMCs indicates coronary artery smooth muscle cells; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell. *P<0.001.

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 1. MARCKS KD impairs dedifferentiated VSMC migration. Human CASMCs were transfected with 100 nM of MARCKS siRNA (KD) or nontargeting, control siRNA (Control). A, At 72 hours post-siRNA treatment, protein expression of MARCKS was decreased by 93%3% compared to cells treated with control (nontargeting) siRNA. B, MARCKS KD significantly attenuated VSMC migration in the wound-healing scratch assay. C, Cell migration was determined by both reduction of the width of the wound. D, The density of cells within the wound. *P<0.05. Scale bars=500 lm. E, Starved CASMCs were stimulated with PDGF (20 ng/mL for 10 minutes), fixed, and stained with anti-cortactin (green) and anti-MARCKS (red) antibodies. White triangles denote the nucleus which stains for neither cortactin nor MARCKS. Lamellipodia, white arrows, formed around the periphery of the cell in the control group, which were seldom detected after MARCKS KD. White arrow heads denote cell nucleus. Scale bars=10 lm; shown are representative images of 3 independent experiments. F, These morphological changes are best appreciated at higher magnification. MARCKS and cortactin colocalized in lamellipodia (white arrows). Scale bar=10 lm. G, Significantly more cells formed lamellipodia in repsonse to PDGF stimulation in the control group compared to the MARCKS KD group. At least 40 cells were counted in each group per experiment. Data are presented as the mean and SE of 3 independent experiments. CASMCs indicates coronary artery smooth muscle cells; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell. *P<0.001.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Migration, Transfection, Control, Expressing, Wound Healing Assay, Staining, Knockdown, Derivative Assay, Small Interfering RNA

Figure 2. MARCKS KD inhibits small GTPase Rac1 and Cdc42 activation and blocks downstream signaling transduction in dedifferentiated VSMCs. A, Human CASMCs were treated with control (Control) siRNA or MARCKS siRNA (MARCKS KD). B, MARCKS siRNA reduced MARCKS protein expression by 94.7%3.1%. *P<0.0001. MARCKS KD did not affect the basal expression of small GTPase Rac1 or Cdc42, or the GTPase effector, PAK1. C, Rac1 and Cdc42 activation was detected in the presence (+PDGF) or absence (PDGF) of PDGF (20 ng/mL for 10 minutes) with the PAK-CRIB pull-down assay, as described in the Materials and Methods section. D, PDGF stimulation increased the activation of Rac1 in control cells, but had no effect after MARCKS KD. E, Similarly, PDGF stimulation increased the activation of Cdc42 in control cells, but also had no effect after MARCKS KD. F and G, MARCKS KD also prevented the activation of the Rac1 and Cdc42 effector, PAK1. Data are presented as the mean and SE of 3 independent experiments. *P<0.05. CASMCs indicates coronary artery smooth muscle cells; CRIB, Cdc42/Rac interactive binding domain; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PAK, p21-activated kinase; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell.

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 2. MARCKS KD inhibits small GTPase Rac1 and Cdc42 activation and blocks downstream signaling transduction in dedifferentiated VSMCs. A, Human CASMCs were treated with control (Control) siRNA or MARCKS siRNA (MARCKS KD). B, MARCKS siRNA reduced MARCKS protein expression by 94.7%3.1%. *P<0.0001. MARCKS KD did not affect the basal expression of small GTPase Rac1 or Cdc42, or the GTPase effector, PAK1. C, Rac1 and Cdc42 activation was detected in the presence (+PDGF) or absence (PDGF) of PDGF (20 ng/mL for 10 minutes) with the PAK-CRIB pull-down assay, as described in the Materials and Methods section. D, PDGF stimulation increased the activation of Rac1 in control cells, but had no effect after MARCKS KD. E, Similarly, PDGF stimulation increased the activation of Cdc42 in control cells, but also had no effect after MARCKS KD. F and G, MARCKS KD also prevented the activation of the Rac1 and Cdc42 effector, PAK1. Data are presented as the mean and SE of 3 independent experiments. *P<0.05. CASMCs indicates coronary artery smooth muscle cells; CRIB, Cdc42/Rac interactive binding domain; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PAK, p21-activated kinase; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Activation Assay, Transduction, Control, Expressing, Pull Down Assay, Binding Assay, Knockdown, Derivative Assay, Small Interfering RNA

Figure 3. Markers of differentiation and MARCKS expression in human CASMCs, rat A10 cells, and rat A7r5 cells. In the present investigation, we used CASMCs and A10 cells for loss-of-function experi- ments. A, We chose these cells because they have lower expression of commonly cited markers of smooth muscle cell differentiation and thus more closely resemble the medial VSMCs in intimal hyperplasia. B, These 2 cell lines had higher consti- tutive expression of MARCKS than A7r5 cells, increasing the power of the KD experiments. A7r5 cells had increased expression of the markers of differentiation and lower constitutive MARCKS expression. Thus, these cells were used in the gain- of-function experiments. CASMCs indicates coro- nary artery smooth muscle cells; MARCKS, myris- toylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; PDGFR, platelet- derived growth factor receptor; SM, smooth muscle; VSMC, vascular smooth muscle cell.

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 3. Markers of differentiation and MARCKS expression in human CASMCs, rat A10 cells, and rat A7r5 cells. In the present investigation, we used CASMCs and A10 cells for loss-of-function experi- ments. A, We chose these cells because they have lower expression of commonly cited markers of smooth muscle cell differentiation and thus more closely resemble the medial VSMCs in intimal hyperplasia. B, These 2 cell lines had higher consti- tutive expression of MARCKS than A7r5 cells, increasing the power of the KD experiments. A7r5 cells had increased expression of the markers of differentiation and lower constitutive MARCKS expression. Thus, these cells were used in the gain- of-function experiments. CASMCs indicates coro- nary artery smooth muscle cells; MARCKS, myris- toylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; PDGFR, platelet- derived growth factor receptor; SM, smooth muscle; VSMC, vascular smooth muscle cell.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Expressing, Cell Differentiation, Derivative Assay

Figure 4. Ectopic expression of wild-type (WT) MARCKS, but not the pseudophosphorylation mutant rescues the migration defects observed in MARCKS KD in dedifferentiated VSMCs. A, Dedifferentiated rat A10 VSMCs were treated with control siRNA (Ctl) or MARCKS siRNA (KD) and cotransfected with plasmids for either pEGFP-N1 vector alone (Vec), WT bovine MARCKS (MARCKS WT), the myristoylation-deficient MARCKS mutant (G2A), the phosphorylation-deficient ED domain MARCKS mutant (S4G), or the pseudophosphorylated ED domain MARCKS mutant (S4D), respectively. B, Cell migration was determined by the wound-healing assay in normal growth media as described in the Materials and Methods section. Cotransfection with WT MARCKS, myristolation-deficient MARCKS, and phosphorylation-deficient MARCKS all rescued migration. The pseudophosphorylation mutant did not rescue migration. Data are presented as the mean and SE of 3 independent experiments and normalized to the control treatment (Ctl+Vec). *P<0.05. C, Activation of the GTPases Rac1 and Cdc42 was determined in the presence of MARCKS KD and cotransfection with WT plasmids and each of the 3 mutant plasmids. D, The WT MARCKS, G2A, and S4G, but not the pseudophosphorylated S4D mutant, rescued both Rac1 and (E) cdc42 activation. Data are presented as the mean and SE of 3 independent experiments normalized to the quiescent condition (PDGF) in control cells (Ctl+Vec). *P<0.05. F, PDGF stimulates MARCKS phosphorylation (Pi-MARCKS) in CASMCs in a time-dependent manner. Maximal MARCKS protein expression was observed after 10 minutes of stimulation. G, MARCKS phosphorylation isalso dependenton the dose ofPDGF.Exposure of starved cells to50 ng/mL yieldedthe greatestphosphorylation of MARCKS at 10 minutes (right panel). CaM indicates calmodulin; ED, effector domain; GFP, green fluorescent protein; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; pEGFP, enhanced green fluorescent protein plasmid; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell; WT, wild type.

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 4. Ectopic expression of wild-type (WT) MARCKS, but not the pseudophosphorylation mutant rescues the migration defects observed in MARCKS KD in dedifferentiated VSMCs. A, Dedifferentiated rat A10 VSMCs were treated with control siRNA (Ctl) or MARCKS siRNA (KD) and cotransfected with plasmids for either pEGFP-N1 vector alone (Vec), WT bovine MARCKS (MARCKS WT), the myristoylation-deficient MARCKS mutant (G2A), the phosphorylation-deficient ED domain MARCKS mutant (S4G), or the pseudophosphorylated ED domain MARCKS mutant (S4D), respectively. B, Cell migration was determined by the wound-healing assay in normal growth media as described in the Materials and Methods section. Cotransfection with WT MARCKS, myristolation-deficient MARCKS, and phosphorylation-deficient MARCKS all rescued migration. The pseudophosphorylation mutant did not rescue migration. Data are presented as the mean and SE of 3 independent experiments and normalized to the control treatment (Ctl+Vec). *P<0.05. C, Activation of the GTPases Rac1 and Cdc42 was determined in the presence of MARCKS KD and cotransfection with WT plasmids and each of the 3 mutant plasmids. D, The WT MARCKS, G2A, and S4G, but not the pseudophosphorylated S4D mutant, rescued both Rac1 and (E) cdc42 activation. Data are presented as the mean and SE of 3 independent experiments normalized to the quiescent condition (PDGF) in control cells (Ctl+Vec). *P<0.05. F, PDGF stimulates MARCKS phosphorylation (Pi-MARCKS) in CASMCs in a time-dependent manner. Maximal MARCKS protein expression was observed after 10 minutes of stimulation. G, MARCKS phosphorylation isalso dependenton the dose ofPDGF.Exposure of starved cells to50 ng/mL yieldedthe greatestphosphorylation of MARCKS at 10 minutes (right panel). CaM indicates calmodulin; ED, effector domain; GFP, green fluorescent protein; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; pEGFP, enhanced green fluorescent protein plasmid; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell; WT, wild type.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Expressing, Mutagenesis, Migration, Control, Plasmid Preparation, Phospho-proteomics, Wound Healing Assay, Cotransfection, Activation Assay, Knockdown, Derivative Assay, Small Interfering RNA

Figure 5. MARCKS KD decreases membrane bound PIP2 in dedifferentiated VSMCs. A, GFP-tagged pleckstrin homology domain of PLC-d (GFP-PH) was used as a live cell biosensor to detect PIP2 levels in human CASMCs. Representative images of cells in each treatment are presented. Scale bars=10 lm. B, Mean intensity values were determined by measuring the pixels along the plasma membrane (Fpm) and in the cytoplasm (Fc). Fluorescence of GFP-PH in plasma membrane is normalized by the mean fluorescence value in the cytoplasm yielding the proportion of PIP2 at the membrane (Fpm/Fc). Cells were treated with MARCKS siRNA (MARCKS KD) or control siRNA (Control). Starved quiescent cells were treated with (+PDGF) or without (PDGF) PDGF (20 ng/mL for 10 minutes). Before exposure to PDGF, significantly more PIP2 localized to the plasma membrane incontrolcells,comparedtoMARCKSKDcells(Fpm/Fc=1.880.42andFpm/Fc=1.170.11,respectively).*P<0.001;n=8.However,afterstimulation with PDGF, there was no significant difference of proportion of PIP2 localized to the plasma membrane (Fpm/Fc=1.080.05 and Fpm/Fc=1.080.11, respectively; n=8). CASMCs indicates coronary artery smooth muscle cells; Fc, fluorescence in cytosol; Fpm, fluorescence in plasma membrane; GFP, green fluorescent protein; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; siRNA, small interfering RNA.

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 5. MARCKS KD decreases membrane bound PIP2 in dedifferentiated VSMCs. A, GFP-tagged pleckstrin homology domain of PLC-d (GFP-PH) was used as a live cell biosensor to detect PIP2 levels in human CASMCs. Representative images of cells in each treatment are presented. Scale bars=10 lm. B, Mean intensity values were determined by measuring the pixels along the plasma membrane (Fpm) and in the cytoplasm (Fc). Fluorescence of GFP-PH in plasma membrane is normalized by the mean fluorescence value in the cytoplasm yielding the proportion of PIP2 at the membrane (Fpm/Fc). Cells were treated with MARCKS siRNA (MARCKS KD) or control siRNA (Control). Starved quiescent cells were treated with (+PDGF) or without (PDGF) PDGF (20 ng/mL for 10 minutes). Before exposure to PDGF, significantly more PIP2 localized to the plasma membrane incontrolcells,comparedtoMARCKSKDcells(Fpm/Fc=1.880.42andFpm/Fc=1.170.11,respectively).*P<0.001;n=8.However,afterstimulation with PDGF, there was no significant difference of proportion of PIP2 localized to the plasma membrane (Fpm/Fc=1.080.05 and Fpm/Fc=1.080.11, respectively; n=8). CASMCs indicates coronary artery smooth muscle cells; Fc, fluorescence in cytosol; Fpm, fluorescence in plasma membrane; GFP, green fluorescent protein; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; siRNA, small interfering RNA.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Membrane, Clinical Proteomics, Fluorescence, Control, Knockdown, Derivative Assay, Small Interfering RNA

Figure 6. Overexpressing MARCKS increases differentiated vascular smooth muscle cell (VSMC) motility. Differentiated rat VSMC A7r5 cells that express low constituative levels of MARCKS protein were transfected with GFP-vector (Vec) or WT bovine MARCKS-GFP (+WT). A, Cells were cotransfected with RFP-PH and with either GFP vector or MARCKS-GFP plasmids. Membrane-associated PIP2 level (Fpm/Fc) was measured and quantified as described in the Materials and Methods section. B, Membrane-associated PIP2 level increased in A7r5 cells overexpressing MARCKS (MARCKS-GFP) compared to control (Vector). *P<0.001, n=8. There was no difference between the 2 treatments in membrane- associated PIP2 after stimulation with PDGF; n=8. Representative images of subcellular location of the RFP-PH biosensor in each treatment are shown on the right panel. Scale bars=10 lm. C, Cell migration increased in A7r5 cells overexpressing MARCKS as determined by the wound- healing assay. *P<0.05; n=3. D, A7r5 Cells were starved for 48 hours before stimulation with PDGF (+PDGF, 10 ng/mL for 10 minutes). Cells were fixed and stained with phalloidin (red) to label the actin cytoskeleton. White arrows denote dorsal ruffles and lamellipodia. Shown are representative images of 3 experiments with independent cell preparations. E, Overexpression of MARCKS promotes dorsal ruffle and lamellipodia formation in differentiated VSMCs. Seventy cells were counted in each group per experiment. *P<0.01. Scale bars=10 lm. F, Activation of the small GTPases Rac1 and Cdc42 was assessed using the PAK-CRIB pull-down assay. G, Overexpression of MARCKS increased Rac1 activity, but not significantly. H, MARCKS overexpression significantly increased Cdc42 activity. Activity was normalized to unstimulated control cells (Vector, PDGF). CRIB indicates Cdc42/Rac interactive binding domain; Fc indicates fluorescence in cytosol; Fpm, fluorescence in plasma membrane; GFP, green fluorescent protein; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PAK, p21- activated kinase; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; RFP, red fluorescent protein; VSMC, vascular smooth muscle cell; WT, wild type. *P<0.05.

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 6. Overexpressing MARCKS increases differentiated vascular smooth muscle cell (VSMC) motility. Differentiated rat VSMC A7r5 cells that express low constituative levels of MARCKS protein were transfected with GFP-vector (Vec) or WT bovine MARCKS-GFP (+WT). A, Cells were cotransfected with RFP-PH and with either GFP vector or MARCKS-GFP plasmids. Membrane-associated PIP2 level (Fpm/Fc) was measured and quantified as described in the Materials and Methods section. B, Membrane-associated PIP2 level increased in A7r5 cells overexpressing MARCKS (MARCKS-GFP) compared to control (Vector). *P<0.001, n=8. There was no difference between the 2 treatments in membrane- associated PIP2 after stimulation with PDGF; n=8. Representative images of subcellular location of the RFP-PH biosensor in each treatment are shown on the right panel. Scale bars=10 lm. C, Cell migration increased in A7r5 cells overexpressing MARCKS as determined by the wound- healing assay. *P<0.05; n=3. D, A7r5 Cells were starved for 48 hours before stimulation with PDGF (+PDGF, 10 ng/mL for 10 minutes). Cells were fixed and stained with phalloidin (red) to label the actin cytoskeleton. White arrows denote dorsal ruffles and lamellipodia. Shown are representative images of 3 experiments with independent cell preparations. E, Overexpression of MARCKS promotes dorsal ruffle and lamellipodia formation in differentiated VSMCs. Seventy cells were counted in each group per experiment. *P<0.01. Scale bars=10 lm. F, Activation of the small GTPases Rac1 and Cdc42 was assessed using the PAK-CRIB pull-down assay. G, Overexpression of MARCKS increased Rac1 activity, but not significantly. H, MARCKS overexpression significantly increased Cdc42 activity. Activity was normalized to unstimulated control cells (Vector, PDGF). CRIB indicates Cdc42/Rac interactive binding domain; Fc indicates fluorescence in cytosol; Fpm, fluorescence in plasma membrane; GFP, green fluorescent protein; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PAK, p21- activated kinase; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; RFP, red fluorescent protein; VSMC, vascular smooth muscle cell; WT, wild type. *P<0.05.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Transfection, Plasmid Preparation, Membrane, Control, Migration, Wound Healing Assay, Staining, Over Expression, Activation Assay, Pull Down Assay, Activity Assay, Binding Assay, Clinical Proteomics, Derivative Assay

Figure 7. Decreased MARCKS expression inhibits intimal hyperplasia formation in vivo. A, MARCKS protein is up-regulated during the formation of intimal hyperplasia in the murine carotid ligation model. Common carotid arteries in WT mice were ligated at the carotid bifurcation and harvested at days 0 (untreated), 7, 14, and 28. Total lysates from 3 arteries (6 lg of total protein) were used to assess phospho-MARCKS (Pi-MARCKS), MARCKS, a- SMA, and b-actin expression at each time point with Western blot analysis. B, Protein expression was normalized to GAPDH. MARCKS expression increases early in the formation of intimal hyperplasia. Maximal MARCKS expression occurs at 14 days. C, The caroitd artery was examined with immunostaining of frozen sections at an early phase of intimal hyperplasia formation (day 7 postligation). Confocal microscopy was used to identify MARCKS (red), a-SMA (green), and the murine endothelial cell marker, CD31 (white). At day 7,theinjuredcarotidarteriesinWTmice had developeda significanthyperplasticlesion.The merged confocal images demonstrate that MARCKS was highly expressed in the injured arteries. MARCKS is localized in the polarized leading edge (white arrows) of invading VSMCs. Scale bars=20 lm. D, MARCKS expression increased in WT mice as a result of carotid ligation (day 14). MARCKS expression increased only slightly after carotid ligation in MARCKS+/ mice (M+/). Rac1 and PAK1 activation were lower in the ligated carotid artery (day 14) of MARCKS+/ mice (M+/) as compared to WT mice. E, Representative VVG-stained sections of ligated carotid artery are shown at day 14 in WT (left) and MARCKS +/ (right) mice. There was a significantly greater hyperplastic response observed in WT mice compared to MARCKS+/ mice (M +/). Scale bars=200 lm. F, Intimal hyperplasia was quantified with intima area/media area ratio, n=3. CD indicates cluster of differentiation; MARCKS, myristoylated alanine-rich protein kinase substrate; PAK, p21- activated kinase; SM, smoothelin; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell; VVG, Verhoeff-Van Gieson; WT, wild type. *P<0.0001.

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 7. Decreased MARCKS expression inhibits intimal hyperplasia formation in vivo. A, MARCKS protein is up-regulated during the formation of intimal hyperplasia in the murine carotid ligation model. Common carotid arteries in WT mice were ligated at the carotid bifurcation and harvested at days 0 (untreated), 7, 14, and 28. Total lysates from 3 arteries (6 lg of total protein) were used to assess phospho-MARCKS (Pi-MARCKS), MARCKS, a- SMA, and b-actin expression at each time point with Western blot analysis. B, Protein expression was normalized to GAPDH. MARCKS expression increases early in the formation of intimal hyperplasia. Maximal MARCKS expression occurs at 14 days. C, The caroitd artery was examined with immunostaining of frozen sections at an early phase of intimal hyperplasia formation (day 7 postligation). Confocal microscopy was used to identify MARCKS (red), a-SMA (green), and the murine endothelial cell marker, CD31 (white). At day 7,theinjuredcarotidarteriesinWTmice had developeda significanthyperplasticlesion.The merged confocal images demonstrate that MARCKS was highly expressed in the injured arteries. MARCKS is localized in the polarized leading edge (white arrows) of invading VSMCs. Scale bars=20 lm. D, MARCKS expression increased in WT mice as a result of carotid ligation (day 14). MARCKS expression increased only slightly after carotid ligation in MARCKS+/ mice (M+/). Rac1 and PAK1 activation were lower in the ligated carotid artery (day 14) of MARCKS+/ mice (M+/) as compared to WT mice. E, Representative VVG-stained sections of ligated carotid artery are shown at day 14 in WT (left) and MARCKS +/ (right) mice. There was a significantly greater hyperplastic response observed in WT mice compared to MARCKS+/ mice (M +/). Scale bars=200 lm. F, Intimal hyperplasia was quantified with intima area/media area ratio, n=3. CD indicates cluster of differentiation; MARCKS, myristoylated alanine-rich protein kinase substrate; PAK, p21- activated kinase; SM, smoothelin; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell; VVG, Verhoeff-Van Gieson; WT, wild type. *P<0.0001.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Expressing, In Vivo, Ligation, Western Blot, Immunostaining, Confocal Microscopy, Marker, Activation Assay, Staining

Phosphorylation of MARCKS is increased in the brains of PKCα-M489V mice. Western blot of lysates of whole brain obtained from nine male and female 3-mo-old wild-type mice (lanes 1–4, males; lanes 5–9, females) or nine male and female genome-edited mice containing a homozygous PKCα-M489V mutation (lanes 10–15, males; lanes 16–18, females). Western blots were probed with antibodies specific to a known PKC phosphorylation site on MARCKS (Ser-159/163) or to total PKCα (Top). Bands were quantified using densitometry and the phospho-MARCKS signal was normalized to total MARCKS signal and PKCα signal was normalized to its β-actin loading control. Normalized data from the depicted Western blot were plotted (Bottom) as average normalized intensity ± SEM (*P < 0.05; n.s., not significantly different using a Student’s t test). Males are indicated in green squares and females in black circles.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Protein kinase Cα gain-of-function variant in Alzheimer’s disease displays enhanced catalysis by a mechanism that evades down-regulation

doi: 10.1073/pnas.1805046115

Figure Lengend Snippet: Phosphorylation of MARCKS is increased in the brains of PKCα-M489V mice. Western blot of lysates of whole brain obtained from nine male and female 3-mo-old wild-type mice (lanes 1–4, males; lanes 5–9, females) or nine male and female genome-edited mice containing a homozygous PKCα-M489V mutation (lanes 10–15, males; lanes 16–18, females). Western blots were probed with antibodies specific to a known PKC phosphorylation site on MARCKS (Ser-159/163) or to total PKCα (Top). Bands were quantified using densitometry and the phospho-MARCKS signal was normalized to total MARCKS signal and PKCα signal was normalized to its β-actin loading control. Normalized data from the depicted Western blot were plotted (Bottom) as average normalized intensity ± SEM (*P < 0.05; n.s., not significantly different using a Student’s t test). Males are indicated in green squares and females in black circles.

Article Snippet: The phospho-MARCKS (sc-12971-R) and the total MARCKS (sc-6454) antibodies were purchased from Santa Cruz Biotechnology. β-Actin antibody was purchased from Sigma-Aldrich (A2228).

Techniques: Western Blot, Mutagenesis