![]() Thirty hours after transfection, the cells were washed twice with PBS and lysed in RIPA buffer (PBS supplemented with 1% Nonidet P-40/0.5% sodium deoxycholate/1 mM PMSF/1 μg/ml aprotinin/1 mM sodium orthovanadate). For immunoprecipitation, 293 cells were transiently transfected by using the FuGENE 6 reagent (Roche Molecular Biochemicals) according to manufacturer protocol. Cell Culture and Immunoprecipitation.ĬOS-1 and 293 cells were maintained at 37☌ with 5% CO 2 in DMEM containing 10% FCS, 50 units/ml penicillin, and 50 μg/ml streptomycin. All constructs were confirmed by DNA sequencing. Mammalian expression vectors for N-terminally FLAG-tagged full-length (MTMR5-WT, amino acids 1–1,930), Δ-coiled coil (MTMR5-Δcoil, amino acids 1–1,640), and Δ-PH (MTMR5-ΔPH, amino acids 1–1,760) MTMR5 proteins were created by inserting cDNA fragments into the 5′ HindIII and 3′ EcoRI sites of pCDNA3.1-NF. ![]() The pGEX- MTMR5 vector was created by inserting the MTMR5 ORF into the 5′- EcoRI and 3′- HindIII sites. The complete MTMR5 ORF was amplified by PCR by using a pMSCVneo- sbf1 as a template ( 30). Vectors for the expression of bacterial recombinant GST-tagged MTMR5 was created by using the pGEX-6P-2 (Amersham Pharmacia). Site-directed mutagenesis was carried out by using a PCR-based procedure ( 29). A PCR product encoding protein fragments that correspond to the MTMR2-ΔPH domain (MTMR2-ΔPH, amino acids 183–643) and the MTMR2-Δcoiled-coil domain (MTMR2-Δcoil, amino acids 1–588) were inserted into the 5′ BglII and 3′ KpnI sites of pEGFP (CLONTECH). Bacterial expression vectors for recombinant His-tagged MTM1 and MTMR2 fusion proteins have also been described ( 7, 11). Vectors for mammalian expression of N-terminally FLAG-tagged MTM1 and MTMR2 and enhanced GFP (EGFP)-tagged MTM1 and MTMR2 fusion proteins have been described ( 7, 11). ![]()
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