RalA and RalB require interaction with RalBP1 but distinct exocyst subunits for regulation of anchorage-independent growth. A, RalA shRNA was used to suppress endogenous RalA expression in SW480 cells. The cells were then infected with pBabe retrovirus vectors encoding WT or effector binding mutant RalA proteins encoded by an shRNA-insensitive cDNA sequence. B, RalB shRNA was used to suppress endogenous RalB expression in SW480 cells. The cells were then infected with pBabe retrovirus vectors encoding WT or effector binding mutant RalB proteins encoded by an shRNA-insensitive cDNA sequence. Blot analyses were done with anti-RalA, anti-RalB and β-actin, to verify equivalent total protein loading. C, RalA and RalB require RalBP1 and the exocyst effector interactions to mediate their roles in CRC cell anchorage-independent growth. D, RalA requires Exo84 and RalB requires Sec5 effector interactions to regulate CRC cell anchorage-independent growth. Data shown are representative of two independent experiments.