Journal: Nucleic Acids Research

Article Title: Alternative splicing and nonsense-mediated mRNA decay regulate mammalian ribosomal gene expression

doi: 10.1093/nar/gki905

Figure Lengend Snippet: Effect of overexpression of r-proteins on the rpL3 splicing pattern. ( A ) Analysis of rpL3 expression in control cells (PC12) and in clones stably transfected with HA-tagged rpL3 cDNA (L3–8 and L3–9) or with HA-tagged rpL12 cDNA (L12). Northern blot hybridizations of total RNA were performed with the indicated probes. Protein samples purified from the same clones were analyzed by western blotting with an HA-tag specific antibody (αHA). ( B ) Quantification of mRNAs shown in panel A. The amounts of HA-mRNA are expressed in arbitrary units after normalization to GAPDH mRNA levels. The mRNA levels of endogenous isoforms were normalized to GAPDH and expressed as a ratio to endogenous levels in untreated control cells. (+), addition of doxycycline; (−), removal of doxycycline. The average of three independent experiments is shown, with SDs.

Article Snippet: Cells were grown to 80–90% confluence and then treated with 100 µg/ml cycloheximide for 4 h or with 20 µM wortmannin for 2 h. The rat PC12 (pheochromocytoma cells) Tet-Off cell line (Clontech) was grown in DMEM supplemented with 5% fetal calf serum (FCS), 10% horse serum, 2 mM l -glutamine and 100 µg/ml G418 (Invitrogen).

Techniques: Over Expression, Expressing, Clone Assay, Stable Transfection, Transfection, Northern Blot, Purification, Western Blot