The distribution of ADAM10 between raft and non-raft membrane regions is not affected by expression of dyn I K44A. A. HEK-M3 cells were transfected with ADAM10, or dyn I K44A, as indicated. Controls received empty vector alone. After 48 h cells were fractionated into detergent-soluble and insoluble fractions. Equal amounts of protein from each fraction were size-fractionated on SDS gels and analyzed by immunoblotting with antibodies to ADAM10, the transferrin receptor (TfR) or the raft protein flotillin 2. B. Total ADAM10 or ADAM10 CTF levels in each fraction were calculated (band intensity × (total fraction volume/volume loaded)) and the amount in the detergent-soluble fraction was divided by the sum of the amounts in both fractions, to determine the fractional distribution of each protein in the non-raft compartment. Results are expressed as means ± SEM from 3 experiments. C. HEK-M3 cells were transfected with N-cadherin and empty vector, or dyn I K44A, fractionated into detergent-soluble and insoluble fractions, and analyzed by immunoblotting with antibodies to the N-cadherin C-terminus. The two halves of each blot were captured from a single image. Several unrelated lanes that appeared on the original images have been omitted for clarity.
Carey et al. BMC Cell Biology 2011 12:20 doi:10.1186/1471-2121-12-20