Fig. 1

Flotillin-2 binds EFR3A in DRMs of HeLa cells. DRM fraction from HeLa cells diluted 1:1 with 2% octyl glucoside in 40 mM Tris–HCl, pH 7.4, 300 mM NaCl and protease inhibitor cocktail (Sigma–Aldrich) were incubated overnight with flotillin-2 Sepharose resin at 4 °C. Then the resin was thoroughly washed with the same buffer and suspended in sample buffer (20% SDS, 50% glycerol, 25 mM EDTA, 250 mM DTT, and 0.25 M Tris pH 6.8), boiled for 5 min, and subjected to SDS-PAGE. A Example of sequence coverage for EFR3A resulting from MS/MS identification of proteins bound to flotillin-2 Sepharose. SDS-PAGE gel fragment containing protein bands larger than 60 kDa was cut out and subjected to MS/MS identification (see also Additional file 1: Figure S1 and Table S1). B Western blotting of samples derived from a pull-down assay. Only bound fraction and unconjugated resin control are shown. Nitrocellulose was probed with an EFR3A antibody (Abnova). C Co-IP results, whole cell lysates of HeLa cells were incubated with 5 μg of anti-FL2 goat antibodies (Abcam) to protein G Dynabeads (Life Technologies) overnight at 4 °C with rotation, according to the manufacturer’s protocol. Nonimmune rabbit IgG (5 μg) was used as a negative control. Input means whole cell lysate proteins, 20 μg protein/well. Immunoprecipitated proteins were eluted in a sample buffer and analyzed by western blotting with rabbit anti-EFR3A antibodies and goat anti-flotillin-1 antibody as a positive control