CD95-Mediated Proton Regulation

Abstract

The Na/H exchanger NHE1 is at the crossroads of a large diversity of signaling pathways, whose activation modifies the cooperative response of the transporter to intracellular H ions. Here we show how the activation of the Na/H exchanger NHE1 by the cleaved ligand of CD95 can be measured. We demonstrate two different methods designed to set intracellular pH at precise values. Then we show how these can be coupled to fast kinetics of lithium transport, which will enable to measure the NHE1 activity like for an enzyme, because they will yield rates of transport.

Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks

“…Fn1 -/-MFs were plated in duplicates in 24-well plates (5X10 4 cells/well). Cells were stimulated with 15 μg/ml of recombinant FN variants, or vehicle buffer (CAPS-saline) for 48 h. Internal pH (pHi) measurement was performed as described (Cophignon et al, 2017). Briefly, cells were incubated with 1 μM BCECF-AM (Thermo Fisher Scientific) for 1 h, rinsed with recovery solution (120 mM sodium chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, 15 mM Hepes), and fluorescence intensity was measured using a multi-well plate spectrophotometer (BioTek Synergy 4, Winooski, VT, USA).…”