Fluor s max

Mitochondria from 3 × 10⁷ cells were extracted as previously described [33 (link)] and suspended in 8 M urea, 4% CHAPS, 65 mM DTE, and 40 mM Tris base and sonicated for 5 s on ice. Following centrifugation at 21000 g, protein concentration was determined by Bradford assay [38 (link)] and 80 μg of total proteins used for 2-dimensional electrophoresis (2DE). Analytical gels were stained with silver nitrate [39 (link)], while semipreparative gels for mass spectrometry analysis were stained with Brilliant Blue R250 (Sigma-Aldrich, USA). Gel images were acquired by Fluor-S MAX multi-imaging system (Bio-Rad Laboratories Italy, Segrate, Italy), and the data were analysed with ImageMaster 2D Platinum software. Gel digestion was carried out according to Shevchenko's protocol [40 (link)] and LC-ESI-MS/MS analysis was performed using a Q-TOF micro™ mass spectrometer (Micromass, UK) as previously described [41 (link)].

Alexa Fluor647-labeled double-stranded palindromic oligonucleotides with (dsApo) or without (dsCpo) the AUREO1 target sequence (Supplementary Table S1) were incubated with various concentrations of PZ mutants, and each reaction mixture was separated in a 5% polyacrylamide gel at 25 ± 1 °C^{15 (link)–17 (link)}. To investigate DNA binding in the L state, each reaction mixture was irradiated with BL and separated by electrophoresis under gel illumination with BL (5 W/m²). The fluorescence signal from the Alexa Fluor647-labeled oligonucleotide of each DNA band was quantified using a Fluor-S MAX image analyzer (Bio-Rad, Hercules, CA, USA) and ImageJ software^{24 (link)}. The fraction of dsApo bound to PZ mutants was determined from the ratio of free dsApo to total dsApo, normalized by the amount of free ssApo, and plotted against the monomer concentrations of PZ mutants applied to the mixture ([monomer]₀). EC₅₀ was estimated by curve fitting to the Hill equation using the maximum of the bound fraction (B_max).

4 mm punch skin biopsies or stratum corneum samples were homogenized in a lysis buffer of TBS/1 M NaCl/ 1% Triton X 10 containing a cocktail of protease inhibitors (Roche, Meylan, France). Lysates were clarified by centrifugation at 10 000 X g at 4°C. Protein determination was performed on subsequent soluble protein supernatants using a BCA assay (Thermo Scientific, Courtaboeuf, France) according to the manufacturer’s instructions. 10 μg aliquots of soluble protein extracts from normal skin biopsies, plantar stratum corneum (PSC) and SC obtained by varnish were separated on Tris-HCl 10–20% gradient SDS-PAGE gels (Bio-Rad, Marnes-la-Coquette, France). Proteins were then transferred to PVDF membranes (Immobilon P, Millipore, Molsheim, France) under standard conditions. Membranes were incubated with primary antibody–a His tagged recombinant monoclonal antibody (Bio-Rad AbDSerotec, Puchheim, Germany) directed against FLG2 N terminal domain (aa2-213) at 1 μg/ml followed by anti His HRP conjugated secondary antibody) (Roche, Meylan, France). For SASPase membranes were incubated with anti–SASPase monoclonal antibody (clone 7H9a105) at 1 μg/ml [7 (link)] followed by an anti-mouse HRP conjugated secondary antibody. Protein bands were revealed by ECL Plus^® (GE Healthcare, Orsay, France) and visualized imaged captured on a gel imager (FluorSmax, Bio-Rad, Marnes-la-Coquette, France).