Pcmv5b flag smurf2 c716a

Murine ChREBP and Mlx expression vectors were kindly gifted by Dr. Howard C. Towle [19 (link)]. In order to induce the overexpression of ChREBP or Smurf2, SV40 MES13 cells were transiently co-transfected with ChREBP or Mlx expression vectors, or transfected with pCMV5B-Flag-Smurf2 wt (Addgene, Cambridge, MA, USA) or pCMV5B-Flag-Smurf2 C716A (catalytically inactive form of Smurf2; Addgene) using Lipofectamine 3000 (Invitrogen), according to the manufacturer’s instructions. For the small interfering RNA (siRNA) transfections, SV40 MES13 cells were plated and transiently transfected with ChREBP siRNA (Santa Cruz Biotechnology), non-specific double-stranded control siRNA (Santa Cruz Biotechnology), Smurf2 siRNA (Bioneer Inc., Daejeon, Korea), Traf4 siRNA (Bioneer Inc.), or scrambled siRNA (Bioneer Inc.) using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions. After 6 h, the medium was replaced with SFM containing 0.1% FAF-BSA and incubated for 16–18 h, following which the cells were stimulated with 10 μM LPA for 3 h or left unstimulated.

The PH-RFP and 2XFYVE-GFP reporter constructs were a generous gift from Dr. Pietro de Camilli (Yale University School of Medicine, New Haven, CT). The PH-RFP and 2XFYVE-GFP constructs contain a PH domain derived from Akt1 showing preferential binding to PI(3,4,5)P3 > PI(3,4,)P2 and a FYVE domain from HRS binding PI(3)P, respectively. pcDNA3-FLAG PTEN was a gift from Jaewhan Song (Addgene plasmid #78777). mCherry-Rab5WT was a gift from Gia Voeltz (Addgene plasmid #49201). mCherry-Rab5DN (S34N) was a gift from Sergio Grinstein (Addgene plasmid #35139). Rab5-GFP lentiviral particles were purchased commercially (AMS Biosciences). mCherry-FKBP-MTM1 was a gift from Tamas Balla (Addgene plasmid #51614). pCMV5B-Flag-SMURF2 C716A was a gift from Jeff Wrana (Addgene plasmid #11747). pRK-Myc-SMURF2 was a gift from Ying Zhang (Addgene plasmid #13678). Ubiquitin WT was a gift from Rachel Klevit (Addgene plasmid #12647). pRK5-HA-Ubiquitin-K48R was a gift from Ted Dawson (Addgene plasmid #17604).

PC9 and A549 cells were cultured in RPMI 1640 medium supplemented with 1% penicillin–streptomycin and 10% FBS. When the cells were 90% confluent, they were transfected with predesigned siRNA oligonucleotides specific for Smurf2 (Life Technologies) using RNAiMAX (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. Scrambled siRNA (Life Technologies) was used as a nonspecific control oligonucleotide. All siRNAs were transfected at a final concentration of 50 nM for the indicated times. For overexpression experiments, the cDNA expression plasmid pCMV5B-Flag-Smurf2 C716A (Addgene) was transiently transfected into cells using Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. The empty vector was transfected as a control.