Retroviral Expression Vectors for SHP2 and PTP1B

Retroviral expression vectors for wild type SHP2 (WT SHP2) and C459E SHP2 (SHP2^C459E) were generated by subcloning human PTPN11 complementary DNA (cDNA) into pMSCV-IRES-EGFP (Clontech). pSUPER-puro-based control shRNA or shPTPN1 retroviral expression vectors were designed to target 5′-CCGCCCAGAGGAGCTATATTC-3′ or 5′-CCGCCCAAAGGAGTTACATTC-3′, respectively, as reported^{49 (link)}. Expression vectors for WT mPTP1B or mPTP1B^C215S were generated by subcloning the appropriate cDNAs into the lentiviral expression vector pFB-neo. Expression vectors for EGFP-fused RAB5A, RAB7A, RAB9A, and RAB11A and pEGFP-WT Dynamin2 and Dynamin2^K44A were purchased from Addgene. A cDNA of mouse catalase that lacks its peroxisome-targeting sequence was purchased from Addgene, and subcloned into pMSCV-IRES-EGFP, adding a start codon followed by a FLAG-tag encoding sequence at the 5′ end. Expression vectors for HyPer3-tk, HyPer3-RAB5, and HyPer3-RAB7 were generated by fusing cDNAs for either the human KRAS C-terminal sequence, human RABA5A or RAB7A (Addgene) to the 3′ end of HyPer3 cDNA, respectively. All constructs were confirmed by DNA sequencing.