Nbp2 47337

Cell extracts were prepared in Laemmli sample buffer. Protein concentrations were determined using Bradford assays. Proteins were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were blocked with 5% dry milk in 0.1% Tween-20 in PBS, incubated with primary antibodies, followed by HRP-conjugated secondary antibodies. The following primary antibodies and dilutions were used: eIF3e (1:1000, C-20, described previously, [52 (link)]), β-actin (1:4000, A5441, Sigma-Aldrich), PARP1 [1:1000, 9532, Cell Signaling Technology (CST)], PARP2 (1:1000, NBP2-47337, Novus Biologicals), PAR (1:1000, ALX-804-220-R100, Enzo Life Sciences), Lys48-linked ubiquitin conjugates (1:1000, 05-1307, Millipore), S6 (1:1000, 2217, CST), pS6 (1:2000, 4858, CST), S6K1 (1:1000, 9202, CST), pS6K1 (1:1000, 9234, CST), TSC2 (1:1000, 4308, CST), 4EBP1 (1:1000, 9452, CST), p4EBP1 (1:1000, 9451, CST). Membranes were developed using ECL Prime reagent and scanned with an ImageQuant LAS500 imaging system (GE Healthcare). Bands of interest were quantified with the ImageQuant TL software (GE Healthcare).

Proteins were extracted utilizing NucleoSpin RNA/Protein extraction kit (Macherey-Nagel, Düren, Germany). Ten micrograms of protein was separated by electrophoresis using Mini-Protean TGX Stain-Free Gels (Bio-Rad). Proteins were transferred onto polyvinyl fluoride membrane and non-specific binding was blocked with 5% non-fat milk in 0.1% Tris-buffered Tween saline buffer. Membranes were incubated with following primary antibodies at +4°C for overnight: anti-human PARP1 rabbit IgG in dilution 1:1,500 (#9532; Cell Signaling Technology, Danvers, MA, USA) and anti-human PARP2 rabbit IgG in dilution 1:1,000 (#NBP2-47337; Novus Biologicals, Littleton, CO, USA). Next, goat anti-rabbit IgG secondary antibody (#111-035-144 in dilution 1:10,000; Jackson ImmunoResearch, West Grove, PA, USA) incubation was performed at room temperature for 1 h. Protein bands were detected utilizing Enhanced Chemiluminescence detection kit (Amersham ECL reagent; GE Healthcare, Barrington, IL) and analyzed with Image Lab Software 6.0 (Bio-rad). Band intensity was normalized to total protein amount in each lane.