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. Author manuscript; available in PMC: 2008 Jan 23.

Published in final edited form as: FEBS Lett. 2006 Dec 14;581(2):223–232. doi: 10.1016/j.febslet.2006.12.015

SR phosphorylation regulates the in vitro binding of PSF to the polypyrimidine tract of the 3’ splice site. (A) Specificity of PSF-pyrimidine (py) tract binding. PSF expressed alone in and purified from E coli was incubated with a labeled RNA oligo (∼1ng) containing the branch point (bp) and py (bp+py) at 30°C for 25 min (lane, 1-4). Specific (lanes 5-9) or non-specific (lanes 10-14) unlabeled competitors were added at varying concentrations to the binding reactions prior to resolution on a 6% native polyacrylamide gel and visualization by phosphoimaging. (B) SR phosphorylation reduces binding. PSF (lanes 1-4) or PSF co-expressed with Dsk1 (PSF^D) (lanes 5-8) or SRPK1 (PSF^S) (lanes 9-12) were incubated separately with the labeled RNA bp+py-containing oligo (∼1ng) at 30°C for 25 min. Samples were resolved and visualized as in(A).

SR phosphorylation regulates the in vitro binding of PSF to the polypyrimidine tract of the 3’ splice site. (A) Specificity of PSF-pyrimidine (py) tract binding. PSF expressed alone in and purified from E coli was incubated with a labeled RNA oligo (∼1ng) containing the branch point (bp) and py (bp+py) at 30°C for 25 min (lane, 1-4). Specific (lanes 5-9) or non-specific (lanes 10-14) unlabeled competitors were added at varying concentrations to the binding reactions prior to resolution on a 6% native polyacrylamide gel and visualization by phosphoimaging. (B) SR phosphorylation reduces binding. PSF (lanes 1-4) or PSF co-expressed with Dsk1 (PSF^D) (lanes 5-8) or SRPK1 (PSF^S) (lanes 9-12) were incubated separately with the labeled RNA bp+py-containing oligo (∼1ng) at 30°C for 25 min. Samples were resolved and visualized as in(A).