Figure 4.

Co-expression and immunoprecipitation of the TSEN complex from HEK cells yields an active complex. (A) FLAG-tagged TSEN constructs were transiently co-expressed and immunoprecipitated, using Anti-FLAG resin, from HEK 293 cells. Isolated samples (IP), soluble cell lysates (input), and the insoluble fraction (pellet) were analyzed by Western blot. TSEN subunits were detected with an anti-FLAG antibody, CLP1 was detected with an anti-GFP antibody, and β-actin was used as a loading control. (B) Denaturing TBE-Urea gels were used to analyze immunoprecipitated samples assayed for endonuclease activity on pre-tRNA-R1, the gel was stained with SYBR Gold. (C) Finally, IP samples were separated on an SDS-PAGE gel and stained to visualize proteins using SYPRO Orange protein stain. TSEN proteins and CLP1 are denoted by ‘*’ in respective lanes.