Figure 5.

UNC119Ab enhances the interactions between RASSF6 and KRASB G12V and between RASSF6 and MDM2. A, B, and C, Myc-tagged, FLAG-tagged, and HA-tagged proteins were expressed in HEK293FT cells as indicated. D, mCherry-MDM2 was expressed in SW480 cells. A, HEK293FT cells were plated at 8 × 10⁵ cells/well in a 6-well plate and transfected with control siRNA (siCont.) or UNC119A siRNA (siUNC119A#1 and #2). 24 h later, cells were transfected with pCIneoMyc-KRASB G12V and pCIneoFHF-RASSF6 (FLAG-RASSF6). 48 h after transfection, immunoprecipitation was performed. UNC119A silencing, which suppressed UNC119A expression (arrowhead), abolished the interaction between RASSF6 and KRASB G12V (white arrows). B, pCIneoMyc-KRASB G12V, pCIneoFHF-RASSF6 (FLAG-RASSF6), and pCIneoFHF-UNC119Ab (FLAG-UNC119Ab) were used. In this experiment, immunoprecipitation was performed 24 h, not 48 h, after transfection. The interaction between RASSF6 and KRASB G12V was barely detectable without UNC119Ab but visible with UNC119Ab (white arrow). The signals were measured by use of ImageJ (numbers under the image). HEK293FT cells (C) and SW480 cells (D) were transfected with control siRNA (siCont.) or KRAS siRNA (siKRAS). 24 h later, transfection was performed with pCIneoFHF-RASSF6 (FLAG-RASSF6), pCIneoMyc-UNC119Ab (Myc-UNC119Ab), pCIneoHAHA-MDM2 (HA-MDM2), or iPS-mCherry-MDM2 (mCherry-MDM2). 48 h after transfection, FLAG-RASSF6 was immunoprecipitated to evaluate the coimmunoprecipitation of HA-MDM2 or mCherry-MDM2. UNC119Ab enhanced the interaction between RASSF6 and MDM2, but KRAS silencing, which suppressed KRAS expression (black arrow), abolished the effect (white arrow). Single asterisks indicate IgG light chains. Three experiments for panels A, B, and C were performed by two members. Three experiments were performed for panel D.