TIP-1 specific antibody blocked the HVGGSSV peptide binding within irradiated tumor.

<p><b>A</b>: Specificity of the TIP-1 antibody as revealed with western blot analysis of whole LLC cell lysate. The endogenous TIP-1 protein (∼14 kD) recognized by the antibody was pointed with arrow. <b>B</b>: Specificity of the TIP-1 antibody as demonstrated in immunofluorescent staining of LLC cells that were transfected with shRNA plasmids. Effect of the TIP-1 targeting shRNA on TIP-1 expression was determined with western blot analysis (upper panel). In the cell staining, the transfected cells were tracked with GFP protein expression from the shRNA plasmids. TIP-1 was stained as red with the TIP-1 antibody. Cell nuclei were stained with DAPI. The LLC cells transfected with the control shRNA that did not affect TIP-1 expression were pointed with arrow head, while the cells transfected with TIP-1 targeting shRNA that abolished TIP-1 expression were pointed with arrows (lower panel). <b>C</b>: ELISA-based <i>in vitro</i> competition assay. Serially diluted antibodies were respectively pre-incubated with the purified GST/TIP-1 proteins (100 ng/well) before the complex was added to the plates coated with the HVGGSSV peptide (50 ng/well). The GST/TIP-1 protein associated to the immobilized HVGGSSV peptide was detected with GST-specific antibody. <b>D</b>: Optical images of LLC tumor-bearing mice that were co-administrated with the Alexa Fluor 750-labeled HVGGSSV peptide and antibodies. LLC tumors in the left hind limbs were irradiated at 5 Gy (pointed with arrows). 200 µg of the TIP-1 antibody, or the control antibody, was injected at 2 hours post the IR treatment, followed by injection of Alexa Fluor 750-labeled HVGGSSV peptide at 4 hours post the IR treatment. Optical images were acquired 24 hours after the peptide injection. The presented data represent three independent experiments.</p

HVGGSSV peptide binds to PDZ domain of the TIP-1 protein.

<p><b>A</b>: Enrichment of phages with selective binding to the HVGGSSV peptide. Phages recovered from the five rounds of screening and those from the original phage library were subjected to selectivity analysis on beads coated with the HVGGSSV peptide or a scrambled peptide control, respectively. Shown are number (PFU) of phages recovered from the beads after the free phages were washed off. 10⁹ PFU of phages were used in this assay. <b>B</b>: Specificity of the TIP-1-expressing phage to the HVGGSSV peptide. <b>C</b>: Diagram of TIP-1 protein shows location of PDZ domain and the critical amino acid (H90) for PDZ ligand binding; <b>D</b>: SDS-PAGE image shows purity of the recombinant TIP-1 proteins and a dysfunctional mutant TIP-1 (H90A). The fusion proteins (∼37 kD) were analyzed along with molecular weight markers. <b>E</b>: Relative association of the purified recombinant proteins to the synthetic peptides was evaluated with ELISA. In these assays, 100 ng of the purified GST/TIP-1 or GST/TIP-1(H90A) proteins were used per wells, all the synthetic peptides were used as 50 ng per well. The mutations in the PDZ binding motif were underlined. Shown are representative data from triplicate experiments. * <i>p</i><0.05, <i>n</i> = 3, the Student's <i>t</i>-test.</p

Radiation induced TIP-1 translocation onto the plasma membrane of the cancer cells.

<p><b>A</b>: Flow cytometric profile of TIP-1 expression on the H460 cell surface. TIP-1 on the cell surface was detected with the TIP-1 antibody 24 hours after radiation treatment, a control IgG was included to demonstrate the antibody specificity. <b>B</b>: Fluorescent staining of the TIP-1 expression (green) on the cell surface of the irradiated H460 cells. DAPI was used for counterstaining. <b>C</b>: Time course study. The H460 cells were irradiated at 5 Gy and then fixed at variable time points after irradiation for flow cytometric analysis of the TIP-1 expression on the cell surface. Percentage of the TIP-1 positive cells was presented. <b>D</b>: The dose-dependence study. The H460 cells were irradiated with variable dose of X-ray, the cells were fixed 24 hours post the irradiation for profiling the TIP-1 expression on the cell surface with flow cytometry. Fold change of the TIP-1 positive cells was calculated by comparison to the untreated cells (counted as 1). <b>E</b>: Western blot analysis of TIP-1 expression within the TIP-1 positive or negative cells that were sorted from the irradiated (5 Gy) H460 cells 24 hours after the irradiation. Relative TIP-1 protein level was normalized to that of the actin control (counted as 1) and shown under the image. <b>F</b>: Flow cytometric profile of TIP-1 expression on the cell surface of LLC, H460 or HUVEC cells. The cells were treated with 5 Gy of X-ray, TIP-1 on the cell surface was profiled 24 hours post the radiation treatment. Fold change of the TIP-1 positive cells was calculated by comparison to the untreated cells (counted as 1). * <i>p</i><0.01, <i>n</i> = 3, the Student's <i>t</i>-test, each was compared to the untreated control, respectively.</p