Figure 2. Localization of αs polybasic mutants.
HEK293 cells were transfected in 6-well plates containing coverslips with 0.4 µg pcDNA3 along with 0.6 µg αspcDNA3 (A), αs5QpcDNA3 (B), or αs4QpcDNA3 (C). 40 hours after transfection cells were fixed with 3.7% formaldehyde and visualized as described under “Experimental Procedures.” D, HEK293 cells were transfected in 6-cm plates with 1.2 µg pcDNA3 along with 1.8 µg pcDNA3 containing the indicated α subunit. Approximately 40 hours after transfection cells were lysed in hypotonic lysis buffer and soluble and particulate cell fractions were separated as described under “Experimental Procedures.” 2.5% of each fraction was resolved on 12% SDS-PAGE and the α subunits were visualized by immunoblotting with the 12CA5 antibody.