Figure 4

The A549 cell line does not express detectable levels of PRMT6 protein. (A) Western blot of cell lysates from the A549, BJAB and HeLa cell lines detected with anti-PRMT6, anti-PRMT1 and anti-β-tubulin antibodies as indicated. (B) Relative expression ratios of PRMT1 and PRMT6 mRNA transcripts in A549 versus HeLa cells. Total RNA were extracted from A549 and HeLa cells before being reverse transcribed into cDNA using random primers. Quantitative PCR was then performed on the cDNA samples using primers specific for PRMT1, PRMT6 and GAPDH transcripts. Relative expression ratios for PRMT1 and PRMT6 were calculated according to the method of [29]. (C) Ectopic Myc-PRMT6 increases the steady state levels of Tat-FLAG protein in A549 cells. Western blotting was performed on A549 cells transfected to express Tat-FLAG with (+) or without (-) Myc-PRMT6. Proteins were detected with anti-FLAG and anti-PRMT6 antibodies, respectively. Loading of cell lysates was normalized for equal amounts of co expressed Renilla luciferase in each sample. (D) A549 cells transfected to express Tat-FLAG with or without Myc-PRMT6 were treated with cycloheximide and harvested at 0, 1, 2, 4, 6 and 8 h post-treatment. Western blotting was performed on total protein-equalized lysates. β-tubulin levels demonstrate equal sample loadings. (E) The Tat-FLAG band intensities in panel D were quantified, and their natural log values were plotted as a function of time. Values for Tat-FLAG co expressed with Myc-PRMT6 are indicated by the black boxes and values for Tat-FLAG expressed alone by the white boxes. The calculated Tat-FLAG protein half-lives are shown in the inset.