Generation of stabile TG2 knockdown NB4 cell line. (A) NB4 cells (control NB4), NB4 cells transduced by random, nontarget shRNA expressing lentivirus (virus control NB4) and NB4 cells transduced by specific shRNA against TG2 expressing lentivirus (TG2 knockdown NB4) were differentiated in the presence of 1μM ATRA for 0, 24, 48, 72, and 96 hours. mRNA expression of TG2 was determined at the indicated time points by real-time Q-PCR, measurements were conducted in triplicates, values are expressed as mean % ± SD of the mean. (B) Control, virus control, and TG2 knockdown NB4 cells were differentiated in the presence of 1μM ATRA for 96 hours. Top panel shows cytosolic activity of TG2 measured at the indicated time point by detecting incorporation of [3H]putrescine into casein. The amount of incorporated [3H]putrescine was determined in a β-counter. Bars depict the means of 3 separate experiments each performed in duplicates. Inset in top panel shows the cytosolic protein levels of TG2 at the indicated time points detected by Western blot. Total protein (25 μg) was loaded in each line, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted and developed with CUB7402 monoclonal antibody against TG2. Bottom panel shows nuclear activity of TG2 measured by detecting incorporation of [3H]putrescine into casein. Inset in bottom panel shows the Western blot analysis of nuclear TG2. TG2 enzyme activity assays and Western blot analyses were performed as described above.