Journal: Nutrients

Article Title: The Protective Effect of Rosmarinic Acid against Unfavorable Influence of Methylparaben and Propylparaben on Collagen in Human Skin Fibroblasts

doi: 10.3390/nu12051282

Figure Lengend Snippet: The influence of rosmarinic acid (RA) on the expression of Collagen Triple Helix Repeat Containing-1 (CTHRC1) and Heat shock protein, 47 kDa (HSP47) at mRNA ( a , c ); and the protein ( b , d ) levels in the fibroblasts treated with methylparaben (MP) and n-propylparaben (PP) ( a , b ), and untreated cells ( c , d ). Expression of genes was assayed by real-time PCR, values represent the mean ± SD of three experiments done in duplicate ( a , c ). Representative gels of Western blotting, the intensity of the bands was quantified by densitometry and normalized to β-actin, values represent the mean (% of control) ± SD of three experiments ( b , d ). Statistically significant differences are marked as: a vs. control, b vs. 0.001% MP and 0.0003% PP, c vs. 0.003% MP and 0.001% PP, d vs. 0.01% MP and 0.003% PP; * p < 0.05; † p < 0.01; ‡ p < 0.001. The values in the bars show a factor of change in comparison to the control (↑, increase; ↓, decrease) or to the respective samples treated with parabens alone (MP and PP) (↗, increase; ↙, decrease).

Article Snippet: Subsequently, they were incubated overnight at 4 °C with: monoclonal antibodies against collagen I (COL1A1), collagen III (COL3A1), total ERK1/ERK2, HSP47, CTHRC1, MMP-1, and MT1-MMP (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a dilution of 1:1000; with monoclonal antibody against Bax (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a dilution of 1:300; monoclonal antibodies against Bcl-xL and MMP-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a dilution of 1:500; monoclonal antibodies against TIMP-1 and TIMP-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a dilution of 1:700; monoclonal antibodies against caspase-3 and phosphorylated ERK1/ERK2 (Cell Signaling Inc., Danvers, MA, USA) at a dilution of 1:1000; and monoclonal antibody against β-actin (Sigma-Aldrich Corp., St. Louis, MO, USA) at a dilution of 1:1000.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Comparison

Journal:

Article Title: Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

doi:

Figure Lengend Snippet: The amino terminus of HTLV-2 p21 is necessary but not sufficient for syncytium formation in BJAB cells. BJAB cells (5 × 105) were transfected with genomic constructs containing chimeric envelopes (shown) as described in Materials and Methods. At day 3 posttransfection, cells were analyzed for syncytia by microscopic analysis. + and − indicate that greater than or less than 10% of the cell population were undergoing cell fusion, respectively. HTLV-2 sequences are depicted by open boxes; HTLV-1 sequences are depicted by dark boxes.

Article Snippet: Western analysis was performed with anti-HTLV-1 gp46 antibody (clone 65/6c2.2.34; Cellular Products Inc., Buffalo, N.Y.) and developed by using the Amersham enhanced chemiluminescence assay.

Techniques: Transfection, Construct

Journal:

Article Title: Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

doi:

Figure Lengend Snippet: HTLV-2 replication in BJAB cells requires sequences at the amino terminus of p21. BJAB cells (5 × 105) were transfected with genomic constructs containing chimeric envelopes as described in Materials and Methods. Every 3 days, the medium was changed by allowing the cells to settle and replacing half of the medium. At 3, 7, 14, and 21 days posttransfection, supernatants were collected and cell debris was removed by centrifugation at 3,000 rpm for 5 min as described in Materials and Methods. The amount of secreted HTLV p24 was quantitated by ELISA as described in Materials and Methods. (A) Clones that induced syncytia in BJAB cells (Fig. ​(Fig.1)1) which included full-length HTLV-2 envelope (II), as well as clones NK, MH, and KB. (B) Clones that did not induce syncytia in BJAB cells (KH, KM, BM, and NKM). (C) Results from the full-length HTLV-2 envelope and the full-length HTLV-1 envelope (NH). The data are representative of three independent experiments. OD550, optical density at 550 nm.

Article Snippet: Western analysis was performed with anti-HTLV-1 gp46 antibody (clone 65/6c2.2.34; Cellular Products Inc., Buffalo, N.Y.) and developed by using the Amersham enhanced chemiluminescence assay.

Techniques: Transfection, Construct, Centrifugation, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal:

Article Title: Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

doi:

Figure Lengend Snippet: The amino terminus of HTLV-2 p21 is required for syncytium formation in HeLa cells. HeLa cells (106) were infected with wild-type vaccinia virus (WR) or VTF7-3 (MOI = 1.0) for 2 h at 37°C. HeLa cells that were infected with WR were transfected with pEM-ClacZβgAn, a plasmid in which the Escherichia coli lacZ gene has been linked to the T7 promoter. Cells that were infected with VTF7-3 were transfected as described in Materials and Methods with vaccinia virus constructs expressing chimeric HTLV envelopes. At 16 h posttransfection, 105 cells from each of the two sources were mixed and scored for blue syncytia as described in Materials and Methods. These data represent the average of two duplicate wells and are representative of two independent experiments. □, HTLV-2; ▪, HTLV-1.

Article Snippet: Western analysis was performed with anti-HTLV-1 gp46 antibody (clone 65/6c2.2.34; Cellular Products Inc., Buffalo, N.Y.) and developed by using the Amersham enhanced chemiluminescence assay.

Techniques: Infection, Transfection, Plasmid Preparation, Construct, Expressing

Journal:

Article Title: Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

doi:

Figure Lengend Snippet: The chimeric HTLV envelopes are expressed efficiently on the cell surface of infected cells. HeLa cells (105) were infected with VTF7-3 for 2 h at 37°C and transfected with vaccinia virus constructs expressing chimeric HTLV envelopes as described in Materials and Methods. Cells were harvested 16 h posttransfection, and 5 × 103 cells were analyzed by flow cytometry for expression of envelope on the cell surface as described in Materials and Methods. The histograms indicate relative cell number (y axis) as a function of relative amount of gp46 on the cell surface (x axis). Mock-infected cells are represented by the white histogram area, and cells transfected with chimeric HTLV envelopes are represented by the dark histogram area.

Article Snippet: Western analysis was performed with anti-HTLV-1 gp46 antibody (clone 65/6c2.2.34; Cellular Products Inc., Buffalo, N.Y.) and developed by using the Amersham enhanced chemiluminescence assay.

Techniques: Infection, Transfection, Construct, Expressing, Flow Cytometry

Journal:

Article Title: Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

doi:

Figure Lengend Snippet: The chimeric HTLV envelopes are similar in the ability to form multimers. HeLa cells (106) were infected with VTF7-3 for 2 h at 37°C and transfected with vaccinia virus constructs expressing chimeric HTLV envelopes as described in Materials and Methods. Cells were harvested 16 h posttransfection and lysed in 500 μl of OGL buffer. (A) Sequence comparison of the amino terminus of HTLV-2 and HTLV-1 p21 proteins. The first 64 aa of HTLV-2 and HTLV-1 p21 are represented. The nonconservative amino acid changes between HTLV-2 and HTLV-1 are boxed. (B) Western analysis of native envelope proteins. Total-cell lysates (20 μl) were separated on a 5 to 20% gradient nondenaturing acrylamide gel with 0.01% SDS in the running buffer as described in Materials and Methods. HTLV envelope was detected by Western blot analysis as described in Materials and Methods. Sizes (in kilodaltons) of the molecular weight standards are indicated on the left; the multimeric forms of HTLV envelope are indicated by the arrow on the right. (C) Western analysis of denatured envelope proteins. Total-cell lysates (20 μl) were heated to 100°C for 5 min and separated on a 5 to 20% gradient nondenaturing acrylamide gel with 0.01% SDS in the running buffer as described in Materials and Methods. HTLV envelope was detected by Western blot analysis as described in Materials and Methods. Sizes of the molecular weight standards are indicated on the left; gp61 is indicated by the arrow on the right.

Article Snippet: Western analysis was performed with anti-HTLV-1 gp46 antibody (clone 65/6c2.2.34; Cellular Products Inc., Buffalo, N.Y.) and developed by using the Amersham enhanced chemiluminescence assay.

Techniques: Infection, Transfection, Construct, Expressing, Sequencing, Western Blot, Acrylamide Gel Assay, Molecular Weight

Journal: Viruses

Article Title: Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

doi: 10.3390/v14040740

Figure Lengend Snippet: Reduction of STLV-1 Env-expressing target cells by infection with rVSV. ( A ) The expression level of STLV-1 Env by Lenti-X 293T cells was analyzed at 24 h after their transfection with an empty vector as a control or STLV-1 Env (Si-2 strain) expression plasmid. These cells were stained with anti-HTLV-1 gp46 MAb or mouse IgG1 MAb as an isotype control and then subjected to flow cytometry. The percentages of STLV-1 gp46-positive cells are presented in each panel. ( B ) Results from an evaluation of the rVSV-mediated killing effects on STLV-1-Env expressing cells are shown. The cells described in ( A ) were mock-infected or infected with G-complemented VSVΔG-AcGFP or VSVΔG-JmGL-AcGFP at an MOI of 1. The numbers of viable cells were counted on days 1, 3, and 5 after infection, and the cell viabilities (% of control) of each group of rVSV-infected Si-2-Env-expressing cells relative to each group of virus-inoculated control cells were calculated. Data obtained from five independent experiments are expressed as the mean ± SD. Asterisks in panel ( B ) represent significant differences versus mock- or VSVΔG-AcGFP-infected cells (** p < 0.001 significance, by two-tailed Student’s t -test with equal variance).

Article Snippet: For the staining of HTLV-1 Env, mouse monoclonal anti-HTLV-1 gp46 Ab [67/5.5.13.1] (Abcam, Cambridge, United Kingdom) [ ], mouse IgG1 kappa monoclonal Ab (isotype) (Abcam, Cambridge, United Kingdom), and goat anti-mouse IgG-PE Ab (Abcam, Cambridge, United Kingdom) were used in accordance with the manufacturer’s protocol.

Techniques: Expressing, Infection, Transfection, Plasmid Preparation, Control, Staining, Flow Cytometry, Virus, Two Tailed Test

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Antibodies Used in This Study

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: In Vitro, In Vivo

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Proteins Cross-Linked to AKAP12 in Normal HSCs

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Sequencing

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Ethanol exposure inhibits the interaction of AKAP12 with the collagen chaperone, heat shock protein 47 (HSP47), in hepatic stellate cells (HSCs). A: Control or ethanol-treated HSCs were immunoprecipitated (IP) with AKAP12 or collagen and then Western blotted (WB) for HSP47 or collagen (Materials and Methods). B: IP blot: Phosphorylated and unphosphorylated fractions of control or ethanol-treated HSCs were purified using Qiagen columns (Materials and Methods) and subjected to immunoprecipitation with HSP47 antibody, followed by Western blotting for AKAP12. Phos-tag blot: AKAP12 phosphorylated (P-AKAP) and unphosphorylated Qiagen fractions from control or ethanol-treated HSCs were compared on Phos-tag gels. Phosphorylated extracellular signal–regulated kinase (p-ERK) was a positive control and is found mainly in the phosphorylated fraction. C: Control or ethanol-fed mouse livers were immunoprecipitated with HSP47 antibody followed by Western blotting for AKAP12. Three experiments are shown. D: Normal or alcoholic hepatitis liver extracts were immunoprecipitated with HSP47 antibody, followed by Western blotting for AKAP12. Experiments were performed in duplicate. Data are expressed as means ± SEM (A–D); n = 5 experiments (A and C); n = 3 experiments (B and D). ∗P < 0.05, ∗∗P < 0.01 versus control; †P < 0.05, ††P < 0.01, and ††††P < 0.001 versus unphosphorylated control; ‡‡‡‡P < 0.001 versus pair-fed control; §§§§P < 0.001 versus normal. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Immunoprecipitation, Western Blot, Purification, Positive Control

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: AKAP12 regulates the ability of collagen to interact with heat shock protein 47 (HSP47). A: Hepatic stellate cells (HSCs) transfected with AKAP12 or negative control siRNA immunoprecipitated (IP) with collagen antibody, followed by HSP47 immunoblotting. B: HSCs transfected with AKAP12 vector or empty vector control were immunoprecipitated with collagen antibody, followed by HSP47 Western blotting (WB). Data are expressed as means ± SEM (A and B). n = 5 experiments (A and B). ∗P < 0.05, ∗∗∗∗P < 0.001 versus negative control; ††P < 0.01, ††††P < 0.001 versus empty vector.

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Transfection, Negative Control, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Summary of mechanisms of action of phospho-AKAP12 in alcoholic liver injury. In normal liver, AKAP12 acts as a scaffold protein for protein kinase C (PKCα) and cyclin-D1 (CCND1). Both hepatocytes and hepatic stellate cells (HSCs) exhibit these functions. Normal HSCs also exhibit specific interaction of AKAP12 with the collagen chaperone, heat shock protein 47 (HSP47), and through this interaction, they might regulate collagen maturation and secretion. During alcoholic liver injury, there is decreased interaction of AKAP12 with PKCα and CCND1 in HSCs because of enhanced AKAP12 phosphorylation and loss of its scaffolding activity. Loss of this scaffolding activity may result in changes in HSC proliferation, cytokinesis, and altered signaling. HSC-specific scaffolding of HSP47 is lost when AKAP12 is phosphorylated on alcohol exposure, and this may facilitate HSP47-collagen chaperoning that may further induce collagen maturation and secretion.

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Scaffolding, Activity Assay