Journal: PLoS ONE
Article Title: Rescue of MHC-1 Antigen Processing Machinery by Down-Regulation in Expression of IGF-1 in Human Glioblastoma Cells
Figure Lengend Snippet: Comparison in expression of TAP, LMP and B-7.1 peptides in parental and pAnti IGF-1 transfected and/or IGF-1R monoclonal antibody (mAb) treated HGB cell lines. 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 Fig 4 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
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, Transfection, Western Blot, SDS Page, Clone Assay, Cell Culture