Fig. 2. RII depletion by siRNA treatment. HeLa cells were incubated (+) or not (-) for 72 hours with an siRNA sequence specific for human RII. Alternatively, they were incubated with a control siRNA specific for the rodent form of this protein. (A) Cells were fixed and processed for immunofluorescence with antibodies specific for RII and C. Cells incubated with siRNA specific for human RII were outlined in the image corresponding to C immunostaining. Bars, 10 µm. (B) Cells were lysed and processed for immunoblotting to detect both RII and C. ß'-COP was detected to normalize the amount of protein loaded. (C) Cells were homogenized and total microsomal membranes were prepared. Membranes were lysed and processed for immunoblotting to detect C. In this case, protein disulfide isomerase (PDI) was used for normalization.