PRAS40 and PRR5-Like Protein Are New mTOR Interactors that Regulate Apoptosis (original) (raw)
Figure 5
PRR5L binds to mTORC2 via SIN1 and/or Rictor but does not affect mTORC2 integrity or kinase activity. A. PRR5L expression in HeLa and HEK293 cells, and PRR5L knock down efficiency.
Top panel: Total RNA was purified from HeLa or HEK293 cells, followed by reverse transcription and PCR with primers corresponding to PRR5L. As a negative control, reverse transcription without the transcriptase enzyme was performed. Endogenous PRR5L is expressed in both cell lines. Bottom panel: HEK293 cells were cotransfected with a GST-PRR5L vector and PRR5L siRNA or control siRNA, and incubated for 48 h. Immunoblots were performed on with antibody against GST or Actin. B. PRR5L binds mTOR via SIN1 and/or Rictor. HEK293 cells were cotransfected with a GST-PRR5L vector and a SIN1 siRNA vector or a control siRNA vectors, and incubated for 4 days. GST pull downs were immunoblotted with the indicated antibodies. GST-PRR5L was detected with an anti-GST antibody. mTOR binding to GST-PRR5L is weaker in the absence of SIN1 and Rictor. C. mTORC2 remains intact in PRR5L knock down cells. HEK293 cells were transfected with PRR5L siRNA or control siRNA and incubated for 48 h. Rictor IPs were immunoblotted with the indicated antibodies. D. mTORC2 readouts are unaltered in PRR5L knock down cells. HEK293 cells were transfected with PRR5L siRNA or control siRNA and incubated for 48 h. Immunoblots were performed on protein extracts with the indicated antibodies. The phosphorylation of Akt S473 and paxillin Y118 is unaltered by PRR5L knock down.