Nik sc 7211

Protein concentration of muscle lysates in passive lysis buffer was determined using the Bradford assay (Bio-Rad Protein Assay Dye Reagent, Bio-Rad Laboratories, Hercules, CA). Fifty to 75 micrograms of protein was denatured and size fractionated on 4–15% SDS-polyacrylamide gels. Proteins were transferred to an Immobilon-FL polyvinylidene fluoride membrane (Millipore, Bedford, PA, USA), blocked in Odyssey blocking buffer (LiCor Biotechnology, Lincoln, NE, USA) and incubated with the appropriate primary antibody according to the manufacturer's instructions. Antibodies included: anti-IKKα/IKK1 (IMG-5477, Imgenex, San Diego, CA, USA), IKKβ/IKK2 (#2684), NF-κB2 (p100/p52; #4882), and p-IκBα (#9246) (Cell Signaling Technology, Danvers, MA, USA), IκBα (sc-371), NF-κB p65 (sc-7151), c-Rel (sc-6363), and NIK (sc-7211) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-GAPDH (G-9545) was used as a loading control (Sigma-Aldrich, St. Louis, MO, USA). Alexa Fluor 680 fluorescent dye-conjugated secondary antibody (Invitrogen) was used for visualization with the Li-Cor Odyssey infrared imaging system described previously [20] (link).

Whole-cell lysates were obtained and analyzed by immunoblot as previously described.^{39 (link)} The following primary antibodies were used: cIAP-1 (1:2000) from R&D Systems; anti-cIAP-2 (1:1000) and anti-IL-6 (ab6672; 1:1000) from Abcam; actin (sc-1615; 1:1000), IκBα (sc-371; 1:1000), NIK (sc-7211; 1:1000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); RIP1 (BD 610458; 1:1000) from BD Biosciences (San Jose, CA, USA); NF-κB2 p100/p52 (#4882; 1:1000) and PARP (#9532; 1:1000) from Cell Signaling Technology (Beverly, MA, USA); GAPDH (MAB374; 1:5000) from EMD Millipore (Temecula, CA, USA).