Figure 2. Suppression of CD91 expression does not reduce GRP94.NTD surface binding to MEF-1 cells.

MEF-1 cells were transiently transfected with either of two CD91-targeting siRNAs (LRP1_1 and LRP1_2) or a vector only (mock) control, and examined for changes in CD91 expression 24 h post-transfection. A. Total RNA samples were isolated and CD91 mRNA knockdown efficiency was determined by RT-PCR. CD91 cDNA from mock-transfected cells displayed linear amplification between PCR cycles 25 and 33 (R² = 0.9974). B. Untransfected (UT), mock-transfected, and siRNA-transfected cells were examined for changes in CD91 mRNA levels using RT-PCR. All samples were tested for DNA contamination using a paired transcriptase-deficient (RT-) reaction, and were examined for off-target effects to 18S rRNA. C & D. MEF-1 cells were transfected with LRP1_2 siRNA or a mock control and analyzed by flow cytometry. CD91 surface expression was determined using 10 μg/mL RAP (C), and GRP94.NTD surface binding was conducted with 25 μg/mL GRP94.NTD (D): unstained cells (thin lines), stained cells (bold lines), mock-transfected cells (grey lines), and LRP1_2 siRNA transfected cells (black lines). Mock-transfected and untransfected cells displayed identical RAP and GRP94.NTD binding (data not shown). The data presented are representative of three independent replicates.