Mino acid composition of relevant regions of constructs used in this study are summarized in Table 1. Plasmids pEGFPc1-MARCH1, pEGFPc1-MARCH9, pCMV8.91, pMD-G and pHR’SIN-cPPT-SGW were provided by Paul Lehner (Cambridge, UK). Plasmids pEGFPc2-MARCH1mut, pEGFPc2-MARCH8wt and MARCH8mut have previously been described [26]. DO and DO constructs were amplified by PCR and cloned into pCDNA3.1 Neo- .Table 1. Composition of DO constructs used in this study.Construct CD8-DO CD8-DO-K225 R DO DOP11V DO DO-K225 R DO-LL242,243 AA DO-K225 R,LL242,243 AA DO-Y227 A DO-K225 R,Y227 A DO-Y227 A,LL242,243 AA DO-K225 R,Y227 A,LL242,243 AAa)Cytoplasmic taila)222 RAQKGYVRTQMSGNEVSRAVLLPQSC247b), c) 222 RAQRGYVRTQMSGNEVSRAVLLPQSC247 216 MGTYVSSVPR225 216 MGTYVSSVPR225 222 RAQKGYVRTQMSGNEVSRAVLLPQSC247 222 RAQRGYVRTQMSGNEVSRAVLLPQSC247 222 RAQKGYVRTQMSGNEVSRAVAAPQSC247 222 RAQRGYVRTQMSGNEVSRAVAAPQSC247 222 RAQKGAVRTQMSGNEVSRAVLLPQSC247 222 RAQRGAVRTQMSGNEVSRAVLLPQSC247 222 RAQKGAVRTQMSGNEVSRAVAAPQSC247 222 RAQRGAVRTQMSGNEVSRAVAAPQSCSubstituted residues in the cytoplasmic tail of DO are in bold and highlighted. Numbering is taken from the mature protein and does not include the signal sequence. c) Single amino acid code is used.b)C2013 WILEY-VCH Verlag GmbH Co. KGaA, Weinheimwww.eji-journal.euMartin Jahnke et al.Eur. J. Immunol. 2013. 43: 1153Flow cytometryFACS analysis was as previously described [33]. Constructs were transfected into HEK 293T cells and cells harvested and analysed 246 h later [26, 34]. Due to residual activity associated with MARCH1-mut and MARCH9-mut constructs inactive MARCH8mut was used as the expression control. This was validated by comparison of surface CD8-reporter expression alone or in the presence of cotransfected MARCH8-mut or GFP. Neither MARCH8-mut or GFP significantly influenced CD8-reporter surface expression. Raji cells were grown in RPMI 1640 supplemented with 10 FCS and 2 mM alanine-glutamine dipeptide. After transduction (36 h), chloroquine (to 60 uM) was added and cells were grown for an additional 12 h. For intracellular staining, cells were fixed in 3 formaldehyde/PBS for 15 min, washed and then permeabilised with 0.2 saponin in FACS buffer. Fc receptors were blocked with 40 human AB serum (Sigma) for 10 min and stained as above but in the presence of 0.Inotuzumab 2 saponin.Coenzyme FO Acquisition of data was performed using an FACScan flow cytometer (BD Biosciences) and data analysed with Summit v4.PMID:23672196 3 software. The expression of surface protein levels was calculated using mean fluorescent intensity (MFI) in the presence of catalytically active E3 ligase as monitored through expression of GFP. Surface expression ( ) = (MFI of cells expressing E3 ligase X 100)/(MFI of cells transfected with march8mut or untransfected cells). Statistical analysis was performed using a two-tailed, unpaired Student’s t-test. Differences were classed as not significant (NS), significant (* p0.05) or highly significant (** p0.01).Conflict of interest: The authors declare no financial or commercial conflict of interest.
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