Hrp conjugated anti rabbit igg sc 2004

Cells were lysed in Laemmli sample buffer containing 5% β-mercaptoethanol and heated at 100°C for 5 min. Protein lysates were then separated on 4–12% gradient Tris-glycine mini protein gels (EC60355BOX; Invitrogen, USA) and transferred onto polyvinylidene difluoride membranes (170-4156; Bio-Rad, USA) using a semidry apparatus (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 and incubated with primary antibodies. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Proteins were detected with enhanced chemiluminescence solution (32106; Thermo Scientific, USA) using an ImageQuant LAS-4000 digital imaging system (GE Healthcare, USA). Primary antibodies used in this study included rabbit anti-ATM (phospho S1981; ab81292; 1:1,000 dilution; Abcam, UK), mouse anti-Lamin A/C antibody (MAB3211; 1:1,000 dilution; Millipore, USA), and rabbit anti-actin (A5060; 1:1000 dilution; Sigma). The secondary antibodies used in this study included HRP-conjugated anti-rabbit IgG (sc-2004; 1:4,000 dilution; Santa Cruz Biotechnology, USA) and HRP-conjugated anti-mouse IgG (sc-2302; 1:4,000 dilution; Santa Cruz Biotechnology). The ratio of progerin to lamin A was quantitated using ImageJ (Collins, 2007 ).