Modification of the detergent extraction procedure eliminates 'nuclear' proteins from detergent-resistant membranes. (A) Cholesterol- and sphingolipid-enriched membranes in LNCaP/MyrAkt1 cells were stained with 0.5 μg/mL Alexa 594-CTxB for 10 min prior to staining with anti-S473-P Akt (1:100) and FITC-conjugated secondary Ab (1:100). Nuclei were counterstained with DAPI prior to imaging. Original magnification, 63×. LNCaP cells stably expressing LacZ or MyrAkt1 were extracted using the conventional (1) or modified (2) detergent extraction procedure. Equal amounts (30 μg for detection of SAFB; 10 μg for other target proteins) of Triton-soluble (TS), Triton-insoluble, octylglucoside-soluble (TI) or nuclear (N) fractions were resolved by SDS-PAGE, transferred to nitrocellulose membranes and (B) stained with Ponceau S or (C) blotted with antibodies to the HA epitope tag, phospho-S473 Akt, PCNA, SAFB, hnRNP K and Giα2.
Adam et al. BMC Cell Biology 2008 9:30 doi:10.1186/1471-2121-9-30