Figure 4.

Tob suppresses the deadenylase activity of Cnot6L in the CCR4‐NOT complex. (a) Green fluorescent protein (GFP)‐Cnot6L was expressed exogenously with or without Flag‐Tob in HEK293T cells. Proteins in the cell lysates were immunoprecipitated with anti‐GFP antibody, and the immunoprecipitates were subjected to immunoblot analysis with anti‐GFP (top panel) or anti‐Flag (bottom panel) antibody. (b) Anti‐GFP immunoprecipitates from HEK293T cells expressing GFP‐Cnot6L alone or GFP‐Cnot6L with Flag‐Tob were incubated in vitro with 5′‐fluorescein‐isothiocyanate‐labeled RNA substrate (RNA‐3′‐20 As) for 60 min. The labeled RNA was then analyzed on a denaturing sequencing gel.