anti icam 2  (R&D Systems)

Journal: Oncogene

Article Title: ICAM-2 confers a non-metastatic phenotype in neuroblastoma cells by interaction with α-actinin

doi: 10.1038/onc.2014.87

Figure Lengend Snippet: (A) Sequence of the cytoplasmic domains of endogenous human ICAM-2 wild type (WT) protein and variants mAB4 and mAB8. The extracellular domain (ED) of the protein contains two immunoglobulin-like domains with six N-glycosylation sites (red lollipops), a 26-amino acid transmembrane domain (TD) and a 26-amino acid cytoplasmic (CD) domain. The N-terminus of the nascent protein contains a 21-amino acid signal peptide (sp) that is not present in the 254 amino acid mature protein. (B) Structures of the 8-mer proposed α-actinin binding domains of ICAM-2 WT, mAB4 and mAB8 were generated using PDB IJ19 and PyMol43. The predicted structure of this domain of ICAM-2 WT is in green. The amino acid side chains of this domain of ICAM-2 mAB4 and mAB8 that have spatial orientations similar to those in the WT protein are also in green.

Article Snippet: Antibodies for IB of ICAM-2, α-actinin, and actin were AF244 (R&D Systems, Minneapolis, MN, USA) and sc-1512 (Santa Cruz Biotech, Santa Cruz, CA, USA), sc-7454R (Santa Cruz), 4968 (Cell Signaling Technology, Danvers, MA, USA), and A5316 (Sigma, St. Louis, MO, USA), respectively.

Techniques: Sequencing, Binding Assay, Generated

Journal: Oncogene

Article Title: ICAM-2 confers a non-metastatic phenotype in neuroblastoma cells by interaction with α-actinin

doi: 10.1038/onc.2014.87

Figure Lengend Snippet: (A) Surface representation of the full-length chicken α-actinin structure (PDB code 1SJJ) reveals a potential binding cavity (arrow) in the cleft between the actin binding domain (ABD, blue) and EF-hand (red) domains. The N-terminus to C-terminus is colored from blue (ABD) to red (EF-hand domain). (B) Structural alignment between full-length chicken α-actinin (red) and the EF-hand domain of human α-actinin (green)(PDB code 1H8B) demonstrated a high confidence alignment (RMSD of 1.47 Å). Structural alignment of the EF-hand domain of chicken α-actinin (red) with EF-hand domain of human α-actinin (residues 823–894, green) complexed to the α-actinin binding domain helix of rabbit titin (known structure, magenta) placed this helix in the putative binding pocket suggested by the structure in “A”. (C) The sequences of the α-actinin binding domains of rabbit titin, murine ICAM-2, and human ICAM-2 have high homology. The secondary structure of the rabbit and murine peptides are indicated as: “h” = helix, “s” = β-sheet and “c” = coil. Amino acid homology comparison is indicated as: “|” = identical; “:” = similar. (D) Modeling reveals a likely high-affinity interaction between the structurally conserved alpha-helix of titin (magenta) and the cytoplasmic domains of murine and human ICAM-2 (by structural and sequence homology) with the EF-hand domain of α-actinin (red). (E) Specific hydrophobic residues of the EF-hand domain of α-actinin and adjacent hydrophobic residues of a helical α-actinin binding structure suggest that ICAM-2 associates directly with α-actinin through hydrophobic interactions. The magenta stick structures represent the “VVAAV” component of the titin helix that is most conserved in ICAM-2.

Article Snippet: Antibodies for IB of ICAM-2, α-actinin, and actin were AF244 (R&D Systems, Minneapolis, MN, USA) and sc-1512 (Santa Cruz Biotech, Santa Cruz, CA, USA), sc-7454R (Santa Cruz), 4968 (Cell Signaling Technology, Danvers, MA, USA), and A5316 (Sigma, St. Louis, MO, USA), respectively.

Techniques: Binding Assay, Comparison, Sequencing

Journal: Oncogene

Article Title: ICAM-2 confers a non-metastatic phenotype in neuroblastoma cells by interaction with α-actinin

doi: 10.1038/onc.2014.87

Figure Lengend Snippet: (A) Immunoblots confirmed that tranfected SK-N-AS cells expressed readily detectable levels of ICAM-2 WT, mAB4 or mAB8. Data in both membranes were generated in the same experiment, but immunoblotted individually. (B) Immunofluorescence staining demonstrated that ICAM-2 WT, mAB4 and mAB8 localized to cell membranes. Scale bar represents 10 μm for all panels. Details of procedures used are in the Methods. (C, D) ICAM-2 WT, but not mAB4 and mAB8, co-precipitated with α-actinin. Immunoprecipitations (IP) were performed using whole cell lysates and a mouse monoclonal antibody to α-actinin (MAB1682, Millipore-Fisher Scientific). The presence of all three types of ICAM-2 proteins in the “input” whole cell lysates (wcl) was confirmed prior to IP.

Article Snippet: Antibodies for IB of ICAM-2, α-actinin, and actin were AF244 (R&D Systems, Minneapolis, MN, USA) and sc-1512 (Santa Cruz Biotech, Santa Cruz, CA, USA), sc-7454R (Santa Cruz), 4968 (Cell Signaling Technology, Danvers, MA, USA), and A5316 (Sigma, St. Louis, MO, USA), respectively.

Techniques: Western Blot, Generated, Immunofluorescence, Staining

Journal: Oncogene

Article Title: ICAM-2 confers a non-metastatic phenotype in neuroblastoma cells by interaction with α-actinin

doi: 10.1038/onc.2014.87

Figure Lengend Snippet: (A) Transfected NB cells that migrated to the distal side of Boyden chamber membranes were imaged after an 18-hour incubation. Representative images were acquired with a Zeiss Axio Observer Z.1 microscope and Zen 2011 Blue software. (B) Migrated cell numbers were determined manually, and analyzed by t-test. ICAM-2 WT, mAB4 and mAB8 inhibited cell motility toward fibronectin compared to cells that expressed no detectable ICAM-2 (Control) (P<0.05*), in an α-actinin-independent manner. (C) ICAM-2 WT, mAB4 and mAB8 inhibited cell migration using collagen I as chemoattractant in an α-actinin-dependent manner (*P=0.0365; **P=0.0018; ***P<0.0001; *P=0.0155; ***P<0.0001).

Article Snippet: Antibodies for IB of ICAM-2, α-actinin, and actin were AF244 (R&D Systems, Minneapolis, MN, USA) and sc-1512 (Santa Cruz Biotech, Santa Cruz, CA, USA), sc-7454R (Santa Cruz), 4968 (Cell Signaling Technology, Danvers, MA, USA), and A5316 (Sigma, St. Louis, MO, USA), respectively.

Techniques: Transfection, Incubation, Microscopy, Software, Migration

Journal: BMC Cancer

Article Title: N-glycosylation of ICAM-2 is required for ICAM-2-mediated complete suppression of metastatic potential of SK-N-AS neuroblastoma cells

doi: 10.1186/1471-2407-13-261

Figure Lengend Snippet: ICAM-2 WT and variants co-precipitated with α-actinin. A ) IP/IB experiments with control cell lines demonstrate the expected association of ICAM-2, α-actinin and actin in lysates from SK-N-ASpIRES.ICAM-2 cells (labeled as WT), but not SK-N-ASpIRESneo cells (neo). Results for these control cell lines were published previously . B ) ICAM-2 glycosylation site variants associated simultaneously with α-actinin and actin. Immunoprecipitations (IP) were performed using whole cell lysates and a mouse monoclonal antibody to α-actinin (MAB1682, Millipore). Following protein separation by electrophoresis, immunoblots (IB) were performed with antibodies to α-actinin (sc-7454, Santa Cruz Biotech), ICAM-2 (AF244, R&D Systems), or actin (4968, Cell Signaling). The presence of ICAM-2 WT and variants in each preparation was confirmed by immunoblot analysis of input preparations (a representative blot is shown in Figure C) and also by immunoblot analysis of the proteins remaining in the supernatant following precipitation (not shown), to confirm the presence of sufficient/excess ICAM-2 protein or variant in each preparation used for immunoprecipitation experiments.

Article Snippet: Immunoblot analyses (Figure C) done with antibody AF244 (R&D Systems) confirmed protein expression in all cell lines except SK-N-ASpIRESneo.

Techniques: Control, Labeling, Glycoproteomics, Electrophoresis, Western Blot, Variant Assay, Immunoprecipitation