Antibodies

2.4.1. Primary Antibodies Anti-IGF-I Antibody (Ab)

A goat polyclonal antiserum (R&D Systems, Wiesbaden, Germany) raised against E.coli-derived recombinant mouse IGF-I was used for the neutralization of IGF-I endogenously produced by cultured rat liver myofibroblasts. Immunoglobulin (Ig) G specific for mouse IGF-I was purified by affinity chromatography.

Anti-IGFBP-2 Ab

A rabbit polyclonal antiserum (Upstate Biotechnology, Lake Placid, NY, USA) raised against bovine IGFBP-2 purified by immunoaffinity chromatography and high-performance liquid chromatography (HPLC) from conditioned medium of Madin Darby Bovine Kidney cells was used for the detection of IGFBP-2 by Western immunoblotting.

Species reactivity: human, mouse, rat, bovine, goat, equine, porcine.

Anti-IGFBP-3 Ab

A rabbit polyclonal antiserum (GroPep, Adelaide, Australia) raised against a synthetic peptide of unique sequence from the central domain of mouse IGFBP-3 was used for the detection of rat IGFBP-3 by Western immunoblotting.

Anti-IGF-II/M6-PR Ab

For the detection of the IGF-II/M6-PR by immunoprecipitation, rabbit polyclonal antiserum raised against a sequence from the extracytoplasmic domain of the IGF-II/M6-PR of rat origin was used. This antibody was kindly provided by Dr. T.Braulke (Dept. of Biochemistry II, University of Goettingen). Species reactivity: rat.

Anti-IGF-IRβ Ab

For the detection of the IGF-IRβ by Western immunoblotting and immunoprecipitation, an affinity purified rabbit polyclonal antiserum raised against a peptide mapping at the carboxy terminus of the IGF-IRβ of human origin was used (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Species reactivity: mouse, rat, human.

Anti-PDGFRβ Ab

An affinity purified rabbit polyclonal antiserum (Santa Cruz Biotechnology) raised against a recombinant protein corresponding to amino acids 958-1106 mapping at the carboxy terminus of the PDGFRβ of human origin was used for the detection of the PDGFRβ by Western immunoblotting and immunoprecipitation. Species reactivity:

mouse, rat, human.

Anti-PDGFRα Ab

For the detection of the PDGFRα by Western immunoblotting and immunoprecipitation, an immunoaffinity purified rabbit polyclonal antibodies raised against a synthetic peptide conjugated with keyhole limpet hemocyanin (KLH) corresponding to amino acids 1035-1053 of human PDGFRα were used (Upstate Biotechnology). Species reactivity:

human, mouse, frog, chicken, rat, zebra fish.

Anti-IRS-1 Ab

For the detection of IRS-1 by Western immunoblotting and immunoprecipitation, an affinity purified rabbit polyclonal antiserum raised against a peptide mapping at the carboxy terminus of IRS-1 of human origin was used (Santa Cruz Biotechnology).

Species reactivity: mouse, rat, human.

Anti-IRS-2 Ab

An affinity purified goat polyclonal antiserum (Santa Cruz Biotechnology) raised against a peptide mapping at the carboxy terminus of IRS-2 of mouse origin was used for the detection of IRS-2 by Western immunoblotting and immunoprecipitation. Species reactivity: mouse, rat, human.

Anti-Gab-1 Ab

For the detection of Gab-1 by Western immunoblotting and immunoprecipitation, a rabbit polyclonal antiserum raised against an 31 residue peptide sequence corresponding to C-terminal residues 664-694 of Gab-1 was used (Upstate Biotechnology). Species reactivity: human, mouse, rat.

Anti-PLCγ1 Ab

An affinity purified rabbit polyclonal antiserum (Santa Cruz Biotechnology) raised against a peptide mapping within the C-terminal domain of PLCγ1 of bovine origin was used for the detection of PLCγ1 by Western immunoblotting and immunoprecipitation.

Species reactivity: mouse, rat, human.

Anti-Phosphotyrosine Ab

A mouse monoclonal antibody (clone PY20) raised against phosphotyrosine (Oncogene, San Diego, CA, USA) was used for the immunovisualization of the

immunoprecipitated proteins phosphorylated on tyrosine residues. This antibody was generated by immunizing BALB/c mice with the phosphotyrosine hapten conjugated to a carrier protein and fusing with NS-1 mouse myeloma cells. Resulting hybridomas were selected for reactivity with the phosphotyrosine hapten by ELISA. Species reactivity: all.

Anti-Phospho-ERK Ab

A mouse phospho-ERK1/2 (Thr202/Tyr204) monoclonal antibody (E10) (Cell Signaling, Beverly, MA, USA) was used to detect endogenous levels of p44 and p42 MAP kinases (ERK1 and ERK2) dually phosphorylated at threonine 202 and tyrosine 204 by Western immunoblotting. This antibody was produced by immunizing mice with a KLH-coupled synthetic phosphopeptide corresponding to residues around Thr202/Tyr204 of human p44 MAP kinase. Species reactivity: human, mouse, rat, hamster, zebra fish.

Anti-Phospho-JNK Ab

A mouse phospho-SAPK/JNK (Thr183/Tyr185) monoclonal antibody (G9) (Cell Signaling, Beverly, MA, USA) purified from ascitic fluid by affinity chromatography was used to detect endogenous levels of p46 and p54 SAPK/JNK dually phosphorylated at threonine 183 and tyrosine 185 by Western immunoblotting. This antibody was generated by immunizing mice with a KLH-coupled synthetic phosphopeptide corresponding to residues around Thr183/Tyr185 of human SAPK/JNK. Species reactivity: human, mouse, rat.

Anti-Phospho-p38 Ab

A mouse phospho-p38 kinase (Thr180/Tyr182) monoclonal antibody (28B10) (Cell Signaling) was used to detect p38 kinase phosphorylated at threonine 180 and tyrosine 182 by Western immunoblotting. This antibody was produced by immunizing mice with a KLH-coupled synthetic phosphopeptide corresponding to residues around Thr183/Tyr185 of human p38 kinase. Species reactivity: human, mouse, rat.

Anti-Fibronectin Ab

A rabbit polyclonal antiserum (Boehringwerke, Marburg, Germany) was used for the detection of fibronectin by Western immunoblotting. Species reactivity: human, rat, mouse.

Anti-Fibulin 2 Ab

An affinity purified goat polyclonal antiserum (Santa Cruz Biotechnology) raised against a peptide mapping near the carboxy terminus of human fibulin-2 was used for the detection of fibulin-2 by Western immunoblotting and immunocytochemistry. Species reactivity: mouse, rat, human.

Anti-Collagen I Ab

A rabbit polyclonal antiserum (Calbiochem, San Diego, CA, USA) raised against type I collagen purified from fetal mouse skin was used for the detection of type I collagen by Western immunoblotting and immunocytochemistry. Species reactivity: human, mouse, rat.

Anti-Collagen IV Ab

A mouse monoclonal antibody (clone IV-4H12) raised against type IV collagen (Oncogene) was used for the detection of this matrix protein by Western immunoblotting. This antibody was generated by immunizing BALB/c mice with a purified type IV collagen protein of human origin and fusing with NS-1 mouse myeloma cells. Species reactivity: human.

Anti-Laminin Ab

A rabbit polyclonal antiserum (Calbiochem, San Diego, CA, USA) was used for the detection of laminin by Western immunoblotting. Species reactivity: rat.

Anti-α-Actin Ab

To detect α-smooth muscle actin, mouse monoclonal antibody (clone 1A4, IgG2a isotype) was used (Sigma, Munich, Germany). This antibody was derived from the hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse. The KLH-coupled N-terminal synthetic decapeptide of α-actin was used as immunogen. This antibody recognizes sarcomeric α-isoform of actin abundantly expressed in skeletal and cardiac muscle.

Anti-β-Actin Ab

For the detection of β-actin, mouse monoclonal antibody (AC-15 clone) raised against N-terminal peptide of β-actin was used (Sigma, Munich, Germany). This antibody

recognizes ubiquitously expressed β-isoform of actin and does not cross-react with sarcomeric α-isoform. Species reactivity: human, bovine, sheep, pig, rabbit, dog, mouse, rat, guinea pig, chicken, carp, fruit fly.

2.4.2. Secondary Antibodies

Peroxidase-Conjugated Swine Anti-Rabbit Immunoglobulins (Ig)

For the detection of a primary antibody of rabbit origin bound to a protein of interest by Western immunoblotting and immunocytochemistry, the secondary horseradish peroxidase-conjugated swine antibodies directed against rabbit immunoglobulins were used (DAKO, Copenhagen, Denmark). Species reactivity: rabbit.

Peroxidase-Conjugated Rabbit Anti-Mouse Ig

For the detection of a primary antibody of mouse origin bound to a protein of interest by Western immunoblotting and immunocytochemistry, the secondary horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins were used (DAKO, Copenhagen, Denmark). Species reactivity: mouse.

Peroxidase-Conjugated Rabbit Anti-Goat Ig

For the detection of a primary antibody of goat origin bound to a protein of interest by Western immunoblotting, the secondary horseradish peroxidase-conjugated rabbit anti-goat immunoglobulins were used (DAKO, Copenhagen, Denmark). Species reactivity:

goat.