FIGURE 2.
Purification of the Drs2p-Cdc50p complex. Digitonin-solubilized membranes prepared from a Δcdc50 mutant strain co-transfected with pCDC50myc and pH2DRS2 were subjected to Ni2+-NTA and α-HA immunoaffinity chromatography, as described under “Experimental Procedures.” A, the following fractions were loaded onto an 8% SDS-PAGE and subjected to silver staining: T, total membranes; SN, solubilized membrane fraction; P, insoluble membrane fraction; FTNTA, flow-through Ni2+-NTA beads; ENTA, Ni2+-NTA eluate; FTHA, flow-through α-HA beads; WHA, washing step; EHA, α-HA bead eluate. B, fractions were analyzed by immunoblotting using α-HA, α-Myc, and α-Lem3p antibodies for detection of pH2-Drs2p, Cdc50p-Myc, and Lem3p, respectively. ENTA and FTHA fractions were diluted 6-fold, and the EHA fraction were diluted 12-fold with respect to the other fractions. C, Ni2+-NTA chromatography of membrane extracts from Δcdc50 mutant strains transfected with pCDC50myc and pH2DRS2 (+) or pCDC50myc alone (−). SN and ENTA fractions were analyzed by immunoblotting as in B.