Figure 4
DHF ricin and IHF ricin show decreased binding to the cell surface and increased degradation in low pH vesicles. (A) HEK293 cells after 3 hours incubation with wild-type ricin, IHF ricin or DHF ricin run on SDS/PAGE under non-reducing conditions. A representative membranes after Western blot with anti-RTA antibodies are shown. Western blots with anti-tubulin antibodies were performed to show equal loading control. Data from three independent experiments are presented. RTA wt is marked as 1, other results are relative to this control. (B) Cells were incubated with wild-type ricin, IHF ricin or DHF ricin for 30 min at 0°C. Binding was measured as described in Methods. A representative membranes after Western blot with anti-RTA antibodies are shown. Western blots with anti-tubulin antibodies were performed to show equal loading control. Data from three independent experiments are presented. RTA wt is marked as 1, other results are relative to this control. (C) Coomasie Blue-stained 12% SDS/PAGE gels showing the stability of 500 ng of wild-type ricin, and modified ricin IHF and DHF after 30 min incubation at 0°C. (D) Cells were treated with or without bafilomycin A1 (0.1 μM) and with wild-type ricin, IHF ricin or DHF ricin. The amounts of ricin that remain in the cell after degradation was analyzed after SDS-PAGE run under non-reducing conditions (see Methods). Representative membranes after Western blot with anti-RTA antibodies are shown. Western blots with anti-tubulin antibodies were performed to show equal loading control. Data from three independent experiments are presented. Ricin wt, ricin IHF and ricin DHF in each graph are marked as 1, other results are relative to this control. All degraded forms of ricin were analyzed. (E) Tyrosine sulfated wild-type ricin sulf-1, and modified ricin sulf-1: IHF or DHF in cells after 3 hours incubation with Na2 35SO4 and further 3 h incubation with the toxins, run on SDS/PAGE under non-reducing conditions. Control experiments revealed equal total sulfation of proteins in cell lysates. A representative autoradiogram is shown.