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. 2013 May 10;5(5):969–982. doi: 10.3390/toxins5050969

Figure 2.

Figure 2

(A) Cells were transfected with a control shRNA vector or two different BiP shRNA constructs either alone or in combination, for three days. Cell lysates were analysed by Western blot using anti-BiP and anti-actin antibodies. (B) Cells transfected with the indicated shRNA were incubated with increasing amounts of ricin in leucine-free medium for 3 h, then washed and incubated with 1 µCi/mL [3H]leucine for 20 min. The incorporation of [3H]leucine was measured and data presented relative to the control (CTRL). Left panel: a representative experiment performed with parallels. Right panel: average IC50 values of 3–4 experiments. Error bars represent standard error of the mean (p < 0.001 for shRNA1 (n = 4) and shRNA1+2 (n = 3) and p = 0.047 for shRNA2 (n = 3)). (C) Cells transfected with the indicated shRNA constructs were incubated with 0.2 mCi/mL35SO42- for 3 h and then with ricin sulf-1 for an additional 3 h. The cells were lysed and ricin was immunoprecipitated and subjected to SDS-PAGE under non-reducing conditions. The amount of ricin A-chain in each sample was quantified and compared to total sulfated ricin in the samples.