His tag

Rabbit antibodies against IKKα (sc-7218, dilution 1:1,000), IKKβ (sc-7607, dilution 1:1,000), NEMO (sc-8330, dilution 1:1,000) and mouse antibody against ubiquitin (sc-8017, dilution 1:1,000) were obtained from Santa Cruz Biotechnology; antibodies against TAK1 (ab109526, dilution 1:1,000), UbcH7 (ab108936, dilution 1:2,000), Ubc13 (ab109286, dilution 1:5,000), RNF8 (ab131221, dilution 1:1,000), p-IKKα S176 (ab138426, dilution 1:500) were from Abcam; antibodies against CYLD (#8462, dilution 1:1,000), p-TAK1 Thr187 (#4536, dilution 1:1,000), p-IKKα/β Ser176/180 (#2697, dilution 1:1,000) and p-IκBα Ser32/36 (#9246, dilution 1:1,000) were from Cell Signaling; antibodies against FLAG-tag (M20008, dilution 1:5,000) and His-tag (M20001, dilution 1:1,000) were from Abmart; antibodies against IκBα and UbcH5c were homemade; and antibody against Tax (mAB, clone Lt-4) was described in Lee et al. [64 (link)].

Proteins were extracted as described previously^{51 (link)}. Equal amounts (30 μg) of protein were separated on a 12% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

Extracted proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat milk and incubated with primary antibodies for β-actin (Proteintech, 81115-1-RR, 1:5000), ALDH1L2 (Proteintech, 21391-1-AP, 1:1000), acetylation (Cell Signaling Technology, 9441S, 1:1000), Flag (MBL, M185–3, 1:5000), SIRT3 (Santa cruz, sc-365175, 1:1000), HA (Proteintech, 51064-2-AP, 1:5000), His-Tag (Abmart, 2A8, 1:1000)