Figure 6. RAF upregulates RFFL expression via MEK and ERK.

A) Knockdown of ARAF, MEK1 and 2, or ERK1 and 2 by siRNAs blocks Gα 12^QL-induced upregulation of RFFL expression. HEK293T cells were cotransfected with the indicated siRNA and the plasmid encoding LacZ or Gα 12^QL. Seventy-two hours later, cells were harvested for q-RT-PCR analysis.

B) Knockdown of ARAF, MEK1 and 2 or ERK1 and 2 by siRNA blocks Gα 12^QL-induced ubiquitination of PRR5L. HEK293T cells were transfected with the indicated siRNA for 48 hrs, followed by transfection with the indicated plasmids. Twenty-hours later, cells were treated with MG132 for 8 hrs before lysis and precipitation with Ni-NTA beads.

C) Overexpression of ARAF, MEK1, or ERK2 stimulates PRR5L ubiquitination. HEK293T cells were transfected with the plasmids as indicated for 24 hrs. The cells were treated with MG132 for 8 hrs before lysis and pull-down with Ni-NTA beads.

D) LPA and Gα12 stimulate ARAF phosphorylation at Ser-299. HEK293T cells were transfected with Gα12^QL or treated with LPA (5 μM, 2hrs), followed by immunoblotting.

E) Gα12 interacts with ARAF. HEK293T cells transfected with the plasmids as indicated. Immunoprecipitation was carried out using a Gα12 or Gα13 antibody in the presence of AMF as described in the “Methods.”

F) Recombinant Gα12 interacts with ARAF, but not CRAF, in a GTP-dependent manner.