Figure 2
Cell surface biotinylation of cell surface proteins identifies endogenous and exogenous TG2. HUVEC confluent monolayers were incubated with GST-TG2/C277A (50 nM) during the last hour of a 5 hr TNFα (10 ng/ml) treatment. Total cell lysates including membrane fractions were isolated after cell surface proteins biotinylation as described under Materials and Methods. (A) Total cell lysates (15 μg) were separated by SDS-PAGE and transferred to PVDF membrane. Blots were developed using Streptavidin-HRP and bands were visualized using ECL chemiluminescence as described under Materials and Methods. (B) Streptavidin bead pull-down of biotinylated proteins from total cell lysates derived from (A) were performed as described under Materials and Methods. The bound biotinylated proteins were eluted from streptavidin-beads by incubating with SDS-PAGE loading buffer with heating at 95 °C for 4-min. Half of the eluted samples were loaded on a SDS-PAGE and transferred to PVDF membrane. Blots were developed using TG2 mouse monoclonal antibody (cub7402) and goat vs mouse IgG conjugated with HRP and bands were visualized using ECL chemiluminescence.
