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. 2017 Aug 16;23:173–184. doi: 10.1016/j.ebiom.2017.08.013

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© 2017 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 1 — Changes in the proteome and cholesterol content of CF neutrophil membranes and lipid rafts.

(a) Neutrophils of ΔF508 homozygous PWCF (CF ∆) were significantly more adherent than cells of HC (P = 0.02, Student's t-test, N = 6). (b) Comparative analysis of proteins extracted from membranes of CF ∆ and HC. The log protein abundance illustrates persistent down-regulation of flotillin-2 (Fl2, P = 0.03), α-enolase (α-Eno, P = 0.01) and talin (Tal, P = 0.006) and up-regulation of CD11b (P = 0.03) on CF ∆ (●) compared to HC (○) (Student's t-test, n = 6 subjects per group). (c) Neutrophil membrane fractions from HC and CF ∆ were subjected to SDS-PAGE and Western blot analysis for flotillin-1 (Fl1), Fl2, annexin-6 (A6), Tal, tubulin (Tub), α-Eno and CD11b. Band intensity for all proteins was quantified by densitometry and normalized to Na⁺/K⁺ ATPase. Significantly decreased levels of Fl1 (P = 0.001), Fl2 (P = 0.01), A6 (P = 0.003), Tal (P = 0.03), Tub (P = 0.007) and α-Eno (P = 0.02), yet increased levels of CD11b (P = 0.02), were detected on CF membranes (n = 6 subjects per group, Student's t-test). (d) Lipid raft fractions were isolated from HC or CF ∆ and subjected to SDS-PAGE analyses. Western blot and densitometry analysis determined significantly decreased Fl1 (P = 0.01) and α-Eno (P = 0.02) and increased CD11b (P = 0.04) in CF ∆ samples (n = 9 subjects per group, Student's t-test). (e) Neutrophil plasma membranes (Mem) and lipid rafts (Rafts) were analysed for cholesterol content using an Amplex ® red cholesterol assay kit. Cholesterol content of samples from CFΔ (P < 0.001 and P = 0.04, respectively, n = 9 subjects per group) were significantly reduced compared to HC membranes (Student's t-test). (f) HC neutrophils (5 × 10⁶ cell/ml) remained untreated or were treated with MβCD and then loaded with calcein-AM dye. LTB₄ (100 nM) and U-75302 were employed as positive and negative controls, respectively. Concentrations of 0.1 mM and 1 mM MβCD (P = 0.03) significantly increased cell adhesion (Student's t-test, n = 5). Each measurement is the mean ± SEM. Coomassie blue stained gel in (d) demonstrates equal protein loading.