Journal: International Journal of Molecular Sciences
Article Title: Cancer Malignancy Is Correlated with Upregulation of PCYT2-Mediated Glycerol Phosphate Modification of α-Dystroglycan
doi: 10.3390/ijms23126662
Figure Lengend Snippet: Effects of enhancement of the GroP modification on cancer cell behaviors. ( A ) TagD overexpression reduced the expression of matriglycans. Cell lysates containing equal amounts of total proteins prepared from HCT116 cells transfected with TagD-Flag-expressing vector or control vector were subjected to immunoblot analysis using IIH6 and AF6868 along with anti-β-actin and anti-Flag antibodies. ( B ) Proliferation assay of HCT116 cells transfected with TagD-expressing vector or control vector at 0, 24, 48, and 72 h. Error bars represent the standard error of the mean (SEM) ( n = 3). ( C ) Transwell assay (scale bar = 600 μm) and ( D ) wound-healing assay (scale bar = 200 μm) for HCT116 cells at 24 h after transfection with TagD-expressing or control vector. ( E ) Wound-healing index of TagD-overexpressing cells and control cells. Individual data points are represented by black dots on the bar graph. Error bars represent the SEM ( n = 3). Significant differences (*) were calculated compared with WT index using two-tailed unpaired Student’s t -test ( p < 0.05).
Article Snippet: The following antibodies were used in this study: anti-Flag (M2) mouse IgG monoclonal antibody (mAb) (Sigma-Aldrich, St. Louis, MO, USA); anti-c-Myc (9E10) mouse IgG mAb (Wako, Osaka, Japan); anti-α-dystroglycan (IIH6C4) mouse IgM mAb (Millipore, Billerica, MA, USA); anti-human dystroglycan (AF6868) sheep IgG polyclonal antibody (pAb) (R&D systems, Minneapolis, MN, USA); anti-PCYT2 (14827) rabbit IgG pAb (Proteintech, Rosemont, IL, USA); anti-β-actin mouse IgG mAb (Sigma-Aldrich); HRP-conjugated anti-mouse IgM mAb (Thermo Fisher Scientific, Waltham, MA, USA); HRP-conjugated anti-mouse IgG mAb (Invitrogen, Carlsbad, CA, USA); HRP-conjugated anti-mouse IgG antibody (Dako, Glostrup, Denmark); HRP-conjugated anti-sheep IgG antibody (Sigma-Aldrich); HRP-conjugated anti-rabbit IgG polyclonal antibody (Cell Signaling, Danvers, MA, USA); mouse monoclonal IgM Ab reactive with GroP-modified α-DG, DG2 [ ].
Techniques: Modification, Over Expression, Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Proliferation Assay, Transwell Assay, Wound Healing Assay, Two Tailed Test
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