Figure 3
Membrane localization and acylation of the amphiphilic helix of M2 fused to RFP. (A) Confocal microscopy: RFP, RFH-AH, RFP-AH-C50S were expressed in BHK21 cells, which were analysed by confocal microscopy. The scale bar is 20 µm. (B) Membrane separation experiment: RFP, RFH-AH, RFP-AH-C50S were expressed in 293 T cells. Cells were lysed and separated into soluble (S) and membranous (M) fractions, which were subjected to blotting with anti-RFP antibodies. 10% of the cytosol preparation and 20% of the membranes were analysed in the blot. (C) Quantification of this and two other independent experiments. The ratio of the density of the M and S bands was calculated, normalized to RFP-AH (= 1). The mean ± SD and the results from the three independent experiments are shown. One-way ANOVA followed by multiple comparison Dunnett test was applied for statistical analysis. *P < 0.05 P = 0.0195, ****P < 0.0001 versus RFP-AH. (D) S-acylation: RFP, RFH-AH, RFP-AH-C50S were expressed in 293 T cells which were lysed 24 h after transfection. To test for protein expression, 10% of the lysate was removed (input). The remainder was divided into two aliquots, one not treated (− HA) and one treated with hydroxylamine (+ HA) to cleave cysteine-bound fatty acids before pulling down proteins with a free SH group. Samples were subjected to Western blotting with antibodies against RFP and, subsequently against flotillin-2, an endogenous acylated protein.
