Kwikquant image analyzer

The proteins in the NEs/whole cell extracts were separated onto a Bio-Rad 4 to 20% gradient Bis-Tris gel, then electrotransferred on a nitrocellulose (0.45 μm pore size; GE Healthcare) membrane using 1× Bio-Rad transfer buffer. The membranes were blocked with 5% w/v skimmed milk in TBST buffer (1× Tris-Buffered Saline, 0.1% Tween 20), then immunoblotted with appropriate antibodies (Pol η [Cell Signaling Technology Cat# 13848S], PNKP, γ-H2AX [ser phos 139 residue; Cell Signaling Technology Cat# 9718S], unmodified H2AX [Cell Signaling Technology Cat# 2595S], p53BP1 [S1778, Cell Signaling Technology Cat# 2675S], unmodified 53BP1 [Santa Cruz Biotechnology Cat# sc 517281], pATM [Santa Cruz Biotechnology Cat# sc 47739], unmodified ATM [Santa Cruz Biotechnology Cat# sc 377293], HDAC2 [GeneTex Cat# 109642]). The membranes were extensively washed with 1% TBST followed by incubation with anti-isotype secondary antibody (Cell Signaling Technology) conjugated with horseradish peroxidase in 5% skimmed milk at room temperature. Subsequently, the membranes were further washed three times (10 min each) in 1% TBST, developed, and imaged using Kwikquant image analyzer and image analysis software (ver 5.2) (Kindle Biosciences).