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/mL³⁵SO₄^2- 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.