Pmscv ires egfp

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.

Retroviral expression vectors for wild‐type SHP2 (WT SHP2) and the mutant SHP2^C459E were generated by subcloning human PTPN11 cDNA into pMSCV‐IRES‐EGFP (Clontech). Ptpn11^fl/fl MEFs expressing WT or mutant SHP2 were generated by retroviral infection, according to the manufacturer's protocol (pMSCV retrovirus system; Clontech), followed by fluorescence‐activated cell sorting (FACS) for EGFP‐positive cells.

The SLC4 human NBCe2-C and NBCe1-A proteins were expressed in HEK-293 cells as follows. Full-length human cDNA for each transporter was cloned into a pMSCV-IRES-EGFP (Clontech, Mountain View, CA) which expresses the transporters under a CMV promoter and also expresses EGFP as a separate protein under an internal ribosome entry site. The cDNA sequence of each of the constructs was verified by DNA sequencing. Use of human material and cell line are approved by UCLA Institutional Biosafety Committee (IBC#111.13.0-r).