Figure 4
Cellular distribution and enzymatic activity of TG2 in response to resveratrol. (A) Representative Western blots showing TG2 protein level in total cell extracts prepared from SHYTG2 and Panc-28 cells. After 48 h treatment in scratch assays, cells near the scratch and away from the scratch were collected separately, and total protein extracts were prepared. Equal amount of proteins were separated on SDS-polyacrylamide gels and Western blots for TG2 were performed. Blots were reprobed with β-actin to indicate loading. (B and C) GTP-binding and Western blots for TG2 in cytoplasmic and membrane protein fractions prepared from SHYTG2 (B) and Panc-28 cells (C). GTP-binding activity of TG2 was performed by UV-irradiating equal amount of proteins (5 μg) in the presence of [α32P]-GTP. UV-irradiated samples were separated by SDS-PAGE, transferred to PVDF membrane and exposed to x-ray films. Similar blots were probed with anti-TG2 antibody for Western blotting. Antibodies against Na+/K+-ATPase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as plasma membrane and cytoplasmic protein markers, respectively. (D, E and F) Bar diagrams representing transamidation activity of TG2 in SHYTG2 and Panc-28 cells. After treatments in scratch assays, total cell lysate of SHYTG2 (D), and cytoplasmic and membrane protein fractions of SHYTG2 and Panc-28 cells (E and F) prepared from cells present near and away from scratch, and transamidation activity was measured by colorimetric assay at 450 nm. Experiments were repeated at least three times. *p < 0.05.