Figure 3

Antisense BCMA RNA affects BCMA protein expression only on translational level. 293T cells were transfected with insert-less vector or vector expressing FhBCMA in the presence of various amounts of p12-ATG expression vector. 48 hours after transfection cells were harvested and the cell suspension split in two. One half (about 10⁷ cells) was lysed in appropriate buffer and 14 μg of the resulting cell lysate was assayed by western blotting using the M2 anti-FLAG monoclonal antibody to test for FhBCMA. We tested that the protein levels were similar in the various samples by western blotting using a rabbit polyclonal anti-actin antibody. The other half of the cell suspension was used to prepare total RNA, which was used for northern blot to assess BCMA and human β-actin levels. 293T cells were transfected with (A) none, (B) 1 μg of insert-less pcDNA3, (C) 100 ng hBCMA, (D) 100 ng p12-ATG, (E) 100 ng hBCMA and 100 ng p12-ATG, (F) 100 ng hBCMA and 900 ng p12-ATG. Except for (A), cells were transfected with total of 1 μg of plasmid using when needed insert-less pcDNA3.