Fusion sl 3500

Finely grinded tissues and cells were extracted in RIPA buffer, and 10 microgram of protein per slot was separated on 12% SDS-PAGE gel, blotted to PVDF membrane, blocked in 5% non-fat milk powder and probed with a rabbit polyclonal antibody against mCherry, which is cross-reactive with other red fluorophore variants, such as tdTomato (Thermo) in 1:1000 dilution. This was followed by a secondary anti-rabbit antibody in 1:10 000 dilution (Sigma-Aldrich). For detection of the endogenous alphaA crystallin an antibody from Santa Cruz (alphaA crystallin, sc-28306) was use at 1: 2000 dilution. For detection an ECL+ kit (GE Healthcare) and an image acquisition system (Vilber Lourmat, Fusion SL 3500) were used.

Fetal fibroblasts and placentas were finely grinded by homogenizer and washed with PBS and lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.2; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.1% aprotinin; 0.1% SDS; 1 mM PMSF). The lysate was clarified by centrifugation (13,000 rpm, 10 min at 4 °C). Equal amounts of proteins from the supernatant was fractionated by 10% SDS-PAGE and electrotransferred onto a PVDF membrane. The blots were blocked in 5% skim milk at 4 °C overnight. In the next day, the membrane was incubated with rabbit anti-klotho antibody (1:1000, Abcam, Cambridge, UK) and rabbit anti-β-actin (1:5000, Abcam) diluted with 5% skim milk Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h. Then membranes were washed three times each for 10 min with TBST incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000) for 1 h. The blots were developed using the Pierce SuperSignal West Pico Chemiluminescent System (Thermo Fisher Scientific). The densities of the immunoblots were scanned with image acquisition system (Fusion SL3500; Vilber Lourmat, Eberhardzell, Germany).

Genomic DNA was isolated by lysing 50 mg of tissue with lysis buffer (100 mM TRIS-HCL pH 8.5; 0.2% SDS; 5 mM EDTA; 200 mM NaCl) and Proteinase K (10 mg/mL) and incubated overnight at 55–60 °C in a shaker. After centrifugation (15 min at 21,000× g) 560 µL of saturated NaCl (5.5 M) was added to 400 µL of the supernatant followed by another centrifugation step (15 min with 14,000 RPM) for protein precipitation. 700 µL of 100% ethanol was added to the supernatant and mixed thoroughly for DNA precipitation. The pellet was washed several times in 70% ethanol, dried at room temperature and resuspended in 50 µL H₂O at 37 °C overnight. DNA was diluted if the concentration was above 2 µg/µL. After digestion with NcoI, the DNA was then electrophoresed, blotted, and hybridized to a DIG-labeled probe against EGFP. After washing, the probe was visualized using a chemiluminescence kit (Roche Diagnostics, Mannheim, Germany) and a Vilber Lourmat Fusion-SL3500 documentation system. Flanking DNA fragments were expected to be >3 kb.