Lamin a c

Cell lysates were mixed with Laemmli buffer (BioRad) with β-mercaptoethanol in a 1:1 volume ratio. Samples were then boiled for 10 min before cooling on ice. Samples were then transferred to a 4–20% Mini PTOTEAN TGX gradient gel (BioRad) and subjected to electrophoresis. Semi-dry transfer of protein to nitrocellulose membrane was done using the Trans-Blot Turbo system from BioRad. After transfer, blots were stored in 1 × PBS, 0.1% Tween-20 wash buffer overnight at 4°C Blots were then blocked in 5% milk dissolved in 1 × PBS, 0.1% Tween-20, for 1 h at room temperature. Rabbit polyclonal antibody to human GDF15 (Santa Cruz Biotechnology, Inc., Dallas, Texas) was used at a 1:500 dilution and incubated for 1 h at room temperature. Lamin A/C (Santa Cruz Biotechnology, Inc.) was used as loading control and rabbit polyclonal antibody against Lamin A/C was used at a 1:2,000 dilution. After primary antibody incubation, blots were washed for 5 min using wash buffer five times with agitation. A HRP conjugated goat anti-rabbit secondary antibody was applied to the membrane in a 1:20,000 dilution and incubated for 1 h at room temperature followed by 5 min wash repeated five times. Membranes were incubated with Supersignal West Dura extended duration substrate (Thermo Scientific, Waltham, MA) and the blot was exposed to Amersham Hyperfilm ECL chemiluminescence film (GE, Pittsburgh, PA).