ab188029 anti mct1 alomone  (Alomone Labs)

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a ) The upper panel shows the trace from size exclusion chromatography of n -dodecyl-β-D-maltose and cholesterol hemisuccinate (DDM/CHS)-solubilised ghost membrane proteins fractionated on a Superdex 200 increase 10/300 column (blue) and for full-length basigin purified from insect cells in CHAPS and exchanged into DDM:CHS (green). The elution volumes of molecular weight standards are indicated above the trace. Fractions collected are demarcated by vertical red dotted lines. The lower panel shows western blots of fractions 2–14. Representative blots for basigin (40–65 kDa, upper panel), PMCA (~138 kDa, upper middle panel), and MCT1 (~45 kDa, lower middle panel) show that all three proteins co-elute predominantly in fractions 7–9. The lower panel shows the equivalent blot for full-length basigin (FL-BSG), related to the green trace. Data shown are representative from n=2 biological replicates. ( b ) Western blot analysis of 2D blue native PAGE/SDS-PAGE separations of human erythrocyte membrane solubilisates before (upper panel) and after pre-incubation with anti-basigin antibody (lower panel). Blot membranes were stained with antibodies specific for PMCA1/4, basigin and MCT1. Markers of apparent complex size indicate the positions of known mitochondrial respiratory chain (super)complexes run in a separate gel lane. Binding of the antibody led to a full size-shift of both PMCA1/4-basigin and MCT1-basigin complexes, whereas no signal of free basigin could be observed in the low molecular weight range, even after overexposure of the blot. Data shown are representative from n=2 biological replicates. ( c ) The left-hand panel shows representative western blot images depicting sequential depletion of PMCA (upper panel) and MCT1 (upper-middle panel). Depletion of both transporters leads to reduced basigin levels, while levels of glycophorin C are unaffected in each fraction, again confirming that basigin is in complex with PMCA or MCT1. The remaining panels show densitometry plots obtained from inverted images of the western blots. Mean integrated band densities are shown with error bars as the standard error of the mean (n=3) and represent technical replicates. ( d ) Bar diagram depicting molecular abundances (abundance norm spec values) of the indicated proteins in depleting affinity purifications with anti-basigin and anti-neuroplastin (NPTN) antibodies from mildly solubilised human erythrocyte membranes as determined by mass spectrometry. The abundance for all proteins was 0 after affinity purification with an IgG control. Data shown are representative from n=8 biological replicates for basigin and n=5 biological replicates for neuroplastin. Figure 1—source data 1. Data associated with . Figure 1—source data 2. Gels and blots associated with .

Article Snippet: All antibodies tested were acquired from commercial sources: anti-TRA-1–85/CD147 antibody (R&D Systems, MAB3195-SP), anti-MCT1 polyclonal antibody (Almone Labs LTD, distributed by Thermo Fisher Scientific, AMT-011), and anti-PMCA polyclonal antibody (Invitrogen, distributed by Thermo Fisher Scientific, PA5-107073).

Techniques: Size-exclusion Chromatography, Membrane, Purification, Molecular Weight, Western Blot, Blue Native PAGE, SDS Page, Incubation, Staining, Binding Assay, Mass Spectrometry, Affinity Purification

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: ( a ) A structural model showing a complex of basigin (blue) and MCT1 (cyan) (based on PDB: 6LYY, ) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, ) has been docked. ( b ) Surface plasmon resonance (SPR) traces after flowing detergent solubilised erythrocyte membrane basigin-rich fractions (red) and membrane fractions depleted for plasma membrane calcium ATPase (PMCA) (pink) and both PMCA and MCT1 (orange) over a PfRH5-coated surface. ( c ) An SPR sensogram showing the binding of a concentration series of basigin-MCT1 (twofold dilutions from 1600 nM) to immobilised PfRH5. Black lines show data and dotted red lines show fitting to a two-state binding model. Data shown are representative from n=3 technical replicates. ( d ) Purified PfRH5 and basigin-MCT1 were assayed for complex formation through size exclusion chromatography using a Superdex 200 increase 10/300 column. The elution profile of basigin-MCT1 alone (blue) and in the presence of PfRH5 (red) are shown. The inset SDS-PAGE gel shows the protein species present in the fractions indicated by stars in the elution trace. ( e ) Growth inhibition assays assessed the effect of antibodies targeting basigin (at 1 μg/ml) and MCT-1 and PMCAs (both at 75 μg/ml) on P. falciparum growth in human blood culture. Five mM EDTA was used as a positive control (100% growth inhibition) while complete media was used as a negative control (0% growth inhibition). Data are the mean and standard deviation of three technical replicates, with a single example from three biological repeats shown. Figure 3—source data 1. Data associated with . Figure 3—source data 2. Gels and blots associated with .

Article Snippet: All antibodies tested were acquired from commercial sources: anti-TRA-1–85/CD147 antibody (R&D Systems, MAB3195-SP), anti-MCT1 polyclonal antibody (Almone Labs LTD, distributed by Thermo Fisher Scientific, AMT-011), and anti-PMCA polyclonal antibody (Invitrogen, distributed by Thermo Fisher Scientific, PA5-107073).

Techniques: SPR Assay, Membrane, Binding Assay, Concentration Assay, Purification, Size-exclusion Chromatography, SDS Page, Inhibition, Positive Control, Negative Control, Standard Deviation

Journal: eLife

Article Title: Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes

doi: 10.7554/eLife.83681

Figure Lengend Snippet: Five different PfRH5-binding monoclonal antibodies were tested for the inhibition of the binding of PfRH5 to ( a ) full-length basigin, ( b ) basigin-PMCA complex, and ( c ) basigin-MCT1 complex. In each case data (n=3) shown are the mean and error bars represent standard error of mean of biological replicates for full-length basigin and basigin-PMCA and technical replicates for basigin-MCT1. ( d ) Structural models showing a complex of basigin (blue) and PMCA (pink) (based on PDB: 6A69, ) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, ) has been docked. Onto this model has been docked either the complex of PfRH5 (yellow) bound to the Fab fragment of R5.016 (red, PDB:6RCV, ) or of 9AD4 (green; 4U0R, ). ( e ) Structural models showing a complex of basigin (blue) and MCT1 (cyan) (based on PDB: 6LYY, ) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, ) has been docked. Onto this model has been docked either the complex of PfRH5 (yellow) bound to the Fab fragment of R5.016 (red, PDB:6RCV, ) or of 9AD4 (green; 4U0R, ). Figure 4—source data 1. Data associated with .

Article Snippet: All antibodies tested were acquired from commercial sources: anti-TRA-1–85/CD147 antibody (R&D Systems, MAB3195-SP), anti-MCT1 polyclonal antibody (Almone Labs LTD, distributed by Thermo Fisher Scientific, AMT-011), and anti-PMCA polyclonal antibody (Invitrogen, distributed by Thermo Fisher Scientific, PA5-107073).

Techniques: Binding Assay, Inhibition