anti tap primary antibody  (Thermo Fisher)

Journal: PLoS ONE

Article Title: Rescue of MHC-1 Antigen Processing Machinery by Down-Regulation in Expression of IGF-1 in Human Glioblastoma Cells

doi: 10.1371/journal.pone.0058428

Figure Lengend Snippet: Demonstration of intracellular IGF-1 levels in the HG-2 cell line by Flowcytometry. Isotype control (non-transfected Cells+mouse IgG FITC); Non-transfected (non-transfected cells+mouse anti-human IGF-1 mAb+goat antimouse IgG FITC); Transfected (transfected cells+mouse antihuman IGF-1 mAb+goat antimouse IgG FITC); Mock transfected (cells transfected with vector minus antisense IGF-1 cDNA+mouse antihuman IGF-1 mAb+goat antimouse IgG FITC). B, Bar graph comparison of IGF-1 expression in transfected and corresponding parental, non-tranfected HGB Cell Lines. Cell lines were established from discarded tumor tissue of Glioblastoma patients. The experiment design was as depicted in legend A of this Fig. % IGF-1 content = %Fs (specific fluorescence) = [Target (Fluorescence mean value) – Control (Fluorescence mean value)]/Target (Fluorescence mean value)×100%. NT = non-transfected, TX = pAnti-IGF-1 transfected. The experiments of Fig 2B were done×3. The paired t-test was used to determine P values. The statistical procedures were performed on the average difference for each cell line before (NT) and after (TX) transfection. The calculated t and associated p-values are given. Grouped comparisons between TX and NT cell lines for IGF-1 from summarized data by two-way ANOVA were statistically significant at p<0.001 (5 cases) or p <0.05 (3 cases).

Article Snippet: Immuno-detection was performed using primary mouse anti-human TAP-1 monoclonal antibody (a gift from Dr. Robert Tampe, Philipps-University of Marburg, Germany) and secondary rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Beverly, MA).

Techniques: Control, Transfection, Plasmid Preparation, Comparison, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Rescue of MHC-1 Antigen Processing Machinery by Down-Regulation in Expression of IGF-1 in Human Glioblastoma Cells

doi: 10.1371/journal.pone.0058428

Figure Lengend Snippet: A, Histogram of fluorescence intensity from isotype control (non-transfected cells+mouse IgG FITC); NT (non-transfected cells+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC); Mock TX (cells transfected with vector devoid of anti IGF-1 cDNA+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC); pAnti IGF-2 TX (cell transfected with vector expressing IGF-2 RNA in antisense orientation+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC); and, pAnti IGF-1 TX (cells transfected with vector pAnti IGF-1+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC). B, Specific fluorescence showing differential expression of HLA-1 from pAnti IGF-1 transfected cells when compared to parental non-transfected cells and other controls (P <0.05). C, Group ANOVA comparison was done by boxplot with Newman-Keuls graphical representation for comparison in expression of HLA-1 in the experimental groups. The data in B was repeated×3.

Article Snippet: Immuno-detection was performed using primary mouse anti-human TAP-1 monoclonal antibody (a gift from Dr. Robert Tampe, Philipps-University of Marburg, Germany) and secondary rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Beverly, MA).

Techniques: Fluorescence, Control, Transfection, Plasmid Preparation, Expressing, Quantitative Proteomics, Comparison

Journal: PLoS ONE

Article Title: Rescue of MHC-1 Antigen Processing Machinery by Down-Regulation in Expression of IGF-1 in Human Glioblastoma Cells

doi: 10.1371/journal.pone.0058428

Figure Lengend Snippet: A, Regulation of TAP-1 peptide in T98G and HG-2 TX cells was determined by Western Blot. Cell lysate was prepared from cells of TX and corresponding NT cell lines pretreated as described in and then subjected to SDS-PAGE and electronically blotted to nitrocellulose membrane. TAP-1 peptide on the membrane was probed by anti-human TAP-1 monoclonal Ab+anti-mouse IgG HRP-linked Ab; and, signal was detected by LumiGLO reagent. Lanes 1, 2 are NT cells from the T98G and HG-2 cell lines respectively. Lane 3, 4 are TX cells from clone b and c of the T98G cell line; Lane 5, 6 are TX cells from clone 5 and 11 of the HG-2 cell line. Re-probed membrane with anti-actin Ab demonstrates presence of the 42 kd actin. Up-regulation in expression of the 70 kd TAP-1 in the two pAnti IGF-1 transfected cell clones were demonstrated in this experiment. B, Up-regulation in TAP-1 and LMP-7 peptides, and, rescue in expression of TAP-2 and LMP-2 peptides following the exogenous addition of 10ug/ml IGF-1R mAb into cell culture medium for 48 hours were demonstrated by Western Blot. The lysates of wild type and IGF-1R antibody treated cells were prepared as described in A. Lanes 1, 2 and 3 represent T98G, HG-2 and HG-9 cell lines cultured in medium with no IGF-1R mAb added, while lanes 4, 5 and 6 are the corresponding respective cell lines cultured in medium with addition of exogenous IGF-1R mAb (10ug/ml), respectively. Rows 1, 2, 3 and 4 represent TAP-1, TAP-2, LMP-2 and LMP-7, respectively. Row 5 demonstrates the rescue in expression of the B-7.1 peptide in IGF-1R mAb treated T98G, HG-2 and HG-9 cell lines when compared to the wild types of the corresponding non-treated cells. The 42 kd band of actin is also shown as an internal control for the quantity of samples loaded. B represents the results from one set of the experiments, that were repeated×2; C, The densitometry analysis for the two sets of similar experiments was obtained by the NIH image J program. The differences in results between the groups with and without IGF-1R mAb in culture media were significant at p<0.05 or p<0.001.

Article Snippet: Immuno-detection was performed using primary mouse anti-human TAP-1 monoclonal antibody (a gift from Dr. Robert Tampe, Philipps-University of Marburg, Germany) and secondary rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Beverly, MA).

Techniques: Western Blot, SDS Page, Membrane, Expressing, Transfection, Clone Assay, Cell Culture, Control

Journal: PLoS ONE

Article Title: Rescue of MHC-1 Antigen Processing Machinery by Down-Regulation in Expression of IGF-1 in Human Glioblastoma Cells

doi: 10.1371/journal.pone.0058428

Figure Lengend Snippet: Variability of TAP-1 levels in pAnti IGF-1 transfected and non-transfected HGB cell lines. Different clones of transfected (TX) and corresponding non-transfected (NT) HGB cell lines, eight from T98G (circles), two from HG-2 (rectangles) and one from HG-3 (triangle), were examined by flow cytometry for the expression of TAP-1. Expression of TAP-1 in TX and NT cell clones were determined using mouse anti-human TAP-1 monoclonal antibody (courtesy of Dr.Robert Tampe) as described in methods. The up-regulated expression of TAP-1 in TX compared to NT clones was significant at the P< 0.05. B, Relative specificity in up-regulation of TAP-1 in pAnti IGF-1 transfected HGB cells. T98G cells were transfected with the vector containing antisense IGF-1 cDNA (TX pAnti IGF-1), with vector containing antisense IGF-2 cDNA (TX pAnti IGF-2), or, with vector containing no antisense sequence (TX pEMT). Transfected cells were comparatively examined by flow cytometry relative to non-transfected T98G cells (NT) for content of TAP-1. The antibody used and flow cytometry analysis were as described in A of this Fig and methods. This experiment was repeated×3. C, TAP-1 expression in comparison of treatments was tested by t-test/ANOVA using Winks SDA software. Boxplot illustrates the comparison in specific fluorescence value among the experimental groups, P<0.001. Represented below the boxplot is the graphical description of Newman-Keuls multiple comparisons.

Article Snippet: Immuno-detection was performed using primary mouse anti-human TAP-1 monoclonal antibody (a gift from Dr. Robert Tampe, Philipps-University of Marburg, Germany) and secondary rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Beverly, MA).

Techniques: Transfection, Clone Assay, Flow Cytometry, Expressing, Plasmid Preparation, Sequencing, Comparison, Software, Fluorescence

Journal: PLoS ONE

Article Title: Rescue of MHC-1 Antigen Processing Machinery by Down-Regulation in Expression of IGF-1 in Human Glioblastoma Cells

doi: 10.1371/journal.pone.0058428

Figure Lengend Snippet: Expression of TAP-1 peptide in T98G cells was down-regulated by addition of IGF-1 to culture medium. TX cells were incubated in absence of serum and in the presence of 50 μM ZnSO 4 and IGF-1 (100 ng/ml) over-night. Lanes 1 and 2 are NT cells from the T98G and HG-2 cell lines respectively. Lane 3 and lane 4 are T98G TX (clone b) and HG-2 TX (clone 5), respectively, into which IGF-1 was added overnight. Lanes 5 and 6 are as in lanes 3 and 4 except without added IGF-1. B. The expression of TAP-1 was examined as a function of time following addition of IGF-1 (100 ng/ml) in TX T98G cells in absence of serum and in the presence of Zn2+. Cells were treated with exogenous IGF-1 for the time periods as depicted. The cell lysates were prepared and subjected to Western Blot analysis as described in methods. The primary antibodies to TAP-1, pStat1 (Tyr701), pStat3 (Tyr705) and Stat3 were also obtained as described. The 70 kd TAP-1, 91 kd pStat1, and 86 kd pStat3 and 86 kd Stat3 peptides produced during the time course were identified by monoclonal antibody used and molecular weights. The data represent one of three sets of similar experimental results. C, Data from Fig 7 B and similar results from two repeat experiments were used for this bar graph. The densitometry quantification was done using the NIH image J program as described in methods.

Article Snippet: Immuno-detection was performed using primary mouse anti-human TAP-1 monoclonal antibody (a gift from Dr. Robert Tampe, Philipps-University of Marburg, Germany) and secondary rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Beverly, MA).

Techniques: Expressing, Incubation, Western Blot, Produced