Skip to main content
. 2016 Aug 26;45(1):417–434. doi: 10.1093/nar/gkw733

Figure 6.

Figure 6.

Differential U2AF35a and U2AF35b expression is U2AF65-dependent. (A) Increased expression of exogenous U2AF35 by free endogenous U2AF65 (enU2AF65). Concentration of the U2AF35 siRNA and plasmids was 30 nM and 200 ng/ml, respectively. Blots were incubated with antibodies against U2AF35 and U2AF65. Knockdown of U2AF35 was associated with a higher mobility U2AF65 fragment (Supplementary Figure S15). (B) U2AF35a and U2AF35b expression depends on their interaction with U2AF65. HEK293 cells were transfected with the indicated Xpress-tagged plasmids and harvested after 48 h. Final concentration of U2AF35 and U2AF65 plasmid DNA in culture media was 130 and 70 ng/ml, respectively. Blots were sequentially exposed to Xpress, U2AF35 and β-actin antibodies. (C and D) Exogenous U2AF35 isoforms are degraded by the proteasome. Blots were successively incubated with antibodies against Xpress (exU2AF35), U2AF35 (enU2AF35), GFP and U2AF65 (enU2AF65 and degU2AF65) (C). A C-terminal degradation product of U2AF65 was described previously in Jurkat cells (78). U2AF expression following addition of lysosomal inhibitor NH4Cl and immunoblotting with the Xpress antibody (D).