In ATRA-treated NB4 cells, both cytosolic and nuclear TG2 are active. (A) Protein-bound N^ϵ-(γ-glutamyl)-lysine cross-links in differentiating NB4 cells. NB4 cells were treated with 1 μM ATRA for 6 days, then cytosolic and nuclear fractions were separated. The N^ϵ-(γ-glutamyl)-lysine cross-link content from each fraction containing 1 to 2 mg protein was determined as described in “Materials and methods.” (B) In situ labeling of proteins of NB4 cells by TG2. Following 1 μM ATRA treatment for 4 days, cells were incubated in the presence of 6 μM 5-(biotinamido)-pentylamine for an additional 12 hours and then separated into cytosolic and nuclear fractions. BPNH₂-labeled proteins were analyzed by SDS-PAGE following immunoblotting with horseradish peroxidase (HRP)–conjugated streptavidin. To detect TG2 in the cytosol, the same blot was probed with monoclonal anti-TG2 antibody. The arrow points to the TG2 bands. Untreated NB4 control cells revealed the endogenous biotinylated proteins. The parallel lanes represent 2 independent experiments.