Myc-mTOR (Addgene plasmid # 1861), pRK5-HA GST RagA 66L (Addgene plasmid # 19300), pRK5-HA GST RagC 75L (Addgene plasmid # 19305) and HA GST PreScission p70 S6K1 (Addgene plasmid # 15511) were gifts from David Sabatini. pcDNA3-FLAG-Rheb-N153T (Addgene plasmid # 19997) was a gift from Fuyuhiko Tamanoi. pcDNA-CaM was a gift from David Yue. TRPML1-HA (Addgene plasmid # 18825) was a gift from Craig Montell. EGFP-Rab7A Q67L (Addgene plasmid # 28049) and EGFP-Rab7A T22N (Addgene plasmid # 28048) were gifts from Qing Zhong.
FLAG-tagged RhebN153T was amplified by PCR and cloned into the EcoRI site of pLVX-AcGFP-N1 vector. GST-tagged 4EBP1 was amplified by PCR and cloned into a pDEST15-based vector. FLAG-tagged CaM was amplified by PCR and cloned into a pGEX6-based vector. TRPML1 was amplified by PCR and cloned into a pEGFP-based vector. All ligations were performed with Infusion Kit (Clontech Laboratories, Inc.) according to the manufacture’s instruction. After sequence verification, these plasmids were used, as described below, in transient cDNA transfections, bacterial protein expression or to produce the lentiviruses needed to generate cell lines stably expressing the proteins.
FLAG-tagged RhebN153T was amplified by PCR and cloned into the EcoRI site of pLVX-AcGFP-N1 vector. GST-tagged 4EBP1 was amplified by PCR and cloned into a pDEST15-based vector. FLAG-tagged CaM was amplified by PCR and cloned into a pGEX6-based vector. TRPML1 was amplified by PCR and cloned into a pEGFP-based vector. All ligations were performed with Infusion Kit (Clontech Laboratories, Inc.) according to the manufacture’s instruction. After sequence verification, these plasmids were used, as described below, in transient cDNA transfections, bacterial protein expression or to produce the lentiviruses needed to generate cell lines stably expressing the proteins.