Proteases and phosphatases inhibitor cocktail

Mouse skin was homogenized in ice cold lysis buffer (50 mm Tris–HCl, pH 7.5, 150 mm NaCl, 0.5% NP40, 20% glycerol) with freshly added proteases and phosphatases inhibitor cocktails (Roche, Vienna, Austria). The homogenate was centrifuged at 15 000 g for 30 min at 4°C. The supernatant was collected and the aliquots were stored at −70°C. The protein content was measured by Bradford assay. Equal amounts of protein were subjected to electrophoresis on 4–12% NuPage gels (Invitrogen, Carlsbad, CA, USA) in 1× MOPS running buffer and then transferred to PVDF membrane. The PVDF membranes were subsequently blocked with 3% non‐fat milk in 1% Tween 20–PBS buffer for 1 h at room temperature. PVDF membranes were incubated with the following primary antibodies: Pso4/Prp19 (SNEV) (1:1000, A300‐102A, Bethyl Lab); γ‐H2AX (1:1000, ab26350; Abcam, Cambridge, UK); p16^INK4a (1:200, SC‐1661; Santa Cruz); MMP‐13 (1:500, ab75606; Abcam); beta‐actin (1:10 000, A5441; Sigma‐Aldrich); and gapdh (1:1000, sc‐25778; Santa Cruz) overnight at 4°C. Thereafter, incubation with secondary antibodies Alexa 680 – (1:10 000; Life Technologies, Carlsbad, CA, USA) or Infrared 800‐conjugated (1:10 000; Lincoln, Nebraska, USA) was performed for 1 h at room temperature. Detection was performed using an Odyssey Infrared scanner, and the optical density of the bands was measured using Image Studio software vs. 2.1 (Licor).

Cells were seeded in 60-mm dishes at 60% confluence and treated according to the experiment. Cells were lysed with RIPA buffer (Tris-HCl 10 mM, pH 7.4; EDTA 5 mM; NaCl 50 mM; deoxycholic acid 1%; triton X-100 1% v/v) in the presence of proteases and phosphatases inhibitor cocktails (Roche, Basilea, Switzerland). Homogenates were centrifuged at 12000 × g for 10 min to eliminate cellular debris, including nuclei. Protein concentrations were measured using the Bradford method [66 (link)] according to the manufacturer’s instructions (Bio-Rad). Protein extracts were denaturated with Laemmli buffer (62.5 mM Tris-base, pH 6.8; 8% glycerol; 2.3% SDS; 0.005% bromophenol blue; 5% 2-ercaptoethanol) for 5 min at 95 °C, then stored at –20 °C.