Journal: Journal of Cell Science
Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes
Figure Lengend Snippet: TMEM16F is required for annexin A2 and A5 cell surface localisation. (A) TMEM16F-knockout (KO) cells have severely reduced annexin A2 and A5 on their surface. Wild-type (WT), matched controls and TMEM-knockout HeLa cells were incubated in versene (EDTA solution) or not (SFM) for 10 min at 37°C before the eluate was collected and analysed for annexin A2 and A5 by western blotting. A representative western blot is shown ( n =4). (B) Expression of mCherry–mTMEM16F rescues lipid movement in TMEM16F-knockout cells. Wild-type and TMEM16F-knockout HeLa cells alone or expressing mCherry–mTMEM16F were treated with ionomycin and analysed for recombinant annexin-A5–Cy5 binding by flow cytometry. A representative experiment is shown ( n =3). (C) Expression of mCherry–mTMEM16F rescues annexin A2 and A5 expression at the cell surface. Annexin A2 and A5 on the cell surface were evaluated through treatment with EDTA and western blotting as described in A. A representative experiment is shown ( n =4). A quantification of cell surface annexin A2 in HeLa transfected or not with mTMEM16F is presented (fold change measured as band intensity [transfected(eluate/lysate)/untransfected(eluate/lysate)]) Results are mean±s.e.m. from n =5 biological replicates; * P
Article Snippet: Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265).
Techniques: Knock-Out, Incubation, Western Blot, Expressing, Recombinant, Binding Assay, Flow Cytometry, Cytometry, Transfection