1. Volume 86, Issue 2, Pages 233-242 (July 1996)
αLβ2 Integrin/LFA-1 Binding to ICAM-1 Induced by Cytohesin-1, a Cytoplasmic Regulatory Molecule 
Waldemar Kolanus, Wolfgang Nagel, Britta Schiller, Lutz Zeitlmann, Samuel Godar, Hannes Stockinger, Brian Seed 
Cell 
Volume 86, Issue 2, Pages (July 1996)
DOI: /S (00)

2. Figure 1 Cytohesin-1 Is a Member of a Novel Family of Proteins
(A) Subdomain structure of cytohesin-1.
(B) Alignment of cytohesin-1 and the cts18.1 fragment.
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3. Figure 2
Northern Blot Analysis of Cytohesin-1 and cts18.1 mRNA Expression
Northern blots of transcripts from various human tissues were hybridized to probes derived from (A) cytohesin-1, (B) cts18.1, and (C) β actin (normalization control). mRNAs had been isolated from peripheral blood leukocytes (lane 1), colon (lane 2), small intestine (lane 3), ovary (lane 4), testis (lane 5), prostate (lane 6), thymus (lane 7), and spleen (lane 8).
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4. Figure 3 Expression of Cytohesin-1 and Subdomains in Jurkat Cells
(A) Schematic diagram of the constructs used in these experiments.
(B) Western blot analysis of wild-type cytohesin-1 and immunoglobulin-fusion proteins derived from recombinant vaccinia viruses. Wild-type cytohesin-1 was immunoprecipitated and detected by anti–cytohesin-1 antibody (lane 1). CIg-fusion proteins were precipitated by Protein A–Sepharose and detected by Protein A–peroxidase (lanes 2–5). Protein bands were visualized by chemiluminescence.
Wild-type cytohesin-1 (lane 1), cIg-control (lane 2), cIg–cytohesin-1 (lane 3), cIg–cyh-SEC7 (lane 4), and cIg–cyh-PH (lane 5).
(C) Demonstration of cIg–cytohesin-1 overexpression. CIg–cytohesin-1 derived from recombinant vaccinia viruses (lane 1) or cellular cytohesin-1 (lane 2) were immunoprecipitated from 107 Jurkat cells, each with the help of anti–cytohesin-1 antibody. CIg–cytohesin-1 is overexpressed at least 10-fold; cellular cytohesin-1 present in the material separated in lane 1 has been competed out by the overexpressed recombinant protein during immunoprecipitation.
(D) Flow cytometric analysis of cytohesin-1 fusion protein expression in permeabilized Jurkat cells using fluorescein isothiocyanate–labeled anti-human IgG antibody.
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5. Figure 4
Effects of Full-Length Cytohesin-1, PH, and SEC7 Domain Wild-Type or Fusion Proteins on Jurkat E6 Cell Adhesion to ICAM-1–Rg and Effects of the Same Proteins on CD18 Surface Expression of Jurkat Cells
(A) Cytohesin-1 wild-type or cIg-fusion proteins induce adhesion of Jurkat cells to ICAM-1–Rg–coated plastic dishes.
(B) Cytohesin-1 or SEC7 domain fusion proteins induce constitutive adhesion of Jurkat cells to ICAM-1–Rg. Expression of the PH domain fusion protein has a dominant-negative effect on the LFA-1–ICAM-1 interaction.
(C) CD18 surface density of Jurkat cells is not changed by cytohesin-1 and subdomain overexpression.
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6. Figure 5
Cytohesin-1 Fusion Protein Expression Has No Effect on α4β1 Integrin Binding to VCAM-1
(A) Schematic of the VCAM-1–Rg expression construct.
(B) Adhesion assay (experiments performed in an analogous fashion to the ones described in Figure 4).
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7. Figure 6
The PH Domain of Cytohesin-1 Specifically Inhibits Jurkat Cell Adhesion to ICAM-1–Rg
(A) Adhesion assay. Various PH domain fusion proteins were tested for their effects on Jurkat binding to ICAM-1, as described in Figure 4.
(B) Western blot analysis of PH domain fusion protein expression.
cIg–cyh-PH (lane 1), cIg–vav-PH (lane 2), cIg–βark-PH (lane 3), cIg–ras-GAP-PH (lane 4).
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8. Figure 7
Biochemical Analysis of the Cytohesin-1–CD18 Interaction and Native Cytohesin-1 Protein Expression in Various Cell Types
(A) Coimmunoprecipitation of CD18 with cytohesin-1 from Jurkat cells.
Starting with whole detergent lysates from Jurkat cells (lane 1), immunoprecipitations were performed with rabbit anti-CD18 antibody (lane 2), rabbit serum (control, lane 3), and anti–cytohesin-1 antibody (lane 4). Monoclonal antibody MEM-48 was used to detect CD18 specifically in the immunoprecipitates. CD18 expression is detected as a cluster of bands migrating between 100 and 120 kDa. No bands were detected when replicas of the blot were stained with isotype-matched control monoclonal antibody IN-05 (mouse IgG1 to insulin).
(B) Cytohesin-1 does not interact with CD29 in Jurkat cells. Analogous experiment to the one shown in Figure 7A.
Total lysate (lane 1), anti-CD29 monoclonal antibody MEM-101A (lane 2), rabbit control serum (lane 3), anti–cytohesin-1 antibody (lane 4). Monoclonal antibody MEM-101A was also used to detect CD29 on the Western blot and is therefore visible as high molecular mass material in lane 2 (nonreducing conditions).
(C) In vitro interaction of histag–cytohesin-1 with CD18cyt–Sepharose. The binding of purified 32P-labeled histag–cytohesin-1 (lane 1, input material) was measured to resin alone (lane 2), to a mixture of two unrelated peptides (lane 3), to a CD29 peptide (lane 4), and to a CD18 peptide (lane 5), each peptide covalently linked to Sepharose. The protein band that corresponds to histag-cytohesin is indicated by the asterisk.
(D) Affinity of histag–cytohesin-1 for CD18 or CD29 cytoplasmic domain peptides coupled to Sepharose. Quantitative analysis was determined as described in Experimental Procedures. Background binding to the matrix had been subtracted from each sample before plotting counts versus input protein concentration.
(E) Immunoprecipitation analysis. Immunoblot of homogenate supernatants from different cell lines using the anticytohesin antibody as precipitating and detecting antibody. As indicated by the arrow, the antibody identifies cytohesin-1 as a 47 kDa cellular protein. The lower molecular mass protein band present in the PBL (peripheral blood lymphocytes) lane is an apparent degradation product of cytohesin-1.
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