Page 66 - 2019_05-HaematologicaMondo-web

M. Ladli et al.
A
B
C
Figure 3. AMPK a1 knockdown disturbs glycophorin A, band 3 expression and phosphorylation of aduccin. (A) Analysis of expression of membrane proteins. Protein extracts from human primary erythroblasts were analyzed by western blot using anti-AMPKa1 antibodies to confirm the knockdown and anti-spectrin a and β, ankyrin, band 3, p-aduccin S726 antibodies. Anti-HSC70 was used as a loading control. AMPKa1, anti-spectrin a, ankyrin, band 3, p-adducin S726 were analyzed on the same western blot, spectrin β on a distinct one (representative western blot at day 8/polychromatic erythroblasts, left panel), the same result was obtained at day 10/polychromatic erythroblasts. The blots were quantified using anti-HSC70 antibodies; values are the mean ± SD of three independent experiments at day 8 and day 10; ns: non-significant, *P<0.05; (right panel). (B) Representative cytometry profile for band 3/a4 integrin gated on GPA-positive cells (left panel) The per- centages of band 3lowa4 integrinhigh and band 3meda4 integrinmed in the shCtrl and shAMPKa1 cell populations are plotted in histograms (right panel). Results are expressed as the mean ± SD of three independent experiments; ns: non-significant, *P<0.05; **P<0.01. (C) Expression of GPA determined by FACS. Representative cytometry profiles are shown for GPA at day 6/basophilic erythroblasts, at day 8/polychromatic erythroblasts and at day 10/orthochromatic erythroblasts. Control isotype: unfilled black for shCtrl, unfilled gray for shAMPKa1; GPA-labeled: filled black for shCtrl, filled gray for shAMPKa1) (left panel). The histogram shows the mean percentage of the GPA fluorescence intensity on the cell surface; 100% represents the fluorescence intensity for shCtrl cells (right panel). Results are expressed as the mean ± SD of three independent experiments; ns: non-significant, *P<0.05; **P<0.01. AMPK: AMP-activated protein kinase; HSC70: heat shock 70kDa protein; d: day; B3: band 3; Baso-E: basophilic erythroblasts; Poly-E: polychromatic erythroblasts; Ortho-E: orthochromatic erythroblasts; GPA: glycophorin A.
912
haematologica | 2019; 104(5)