α p iκb α

Nuclear or cytoplasmic extracts were prepared by the use of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Protein concentration was determined by a DC protein assay kit (Bio-Rad, Richmond, CA, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore, Bedford, MA, USA). These membranes were probed with anti-WDR35 peptide antibody (amino acids 459–473, 1∶500), which was designed, produced, and purified by Medical & Biological Laboratories (Nagoya, Japan), or with antibodies against c-Jun, phospho-c-Jun (Ser73) (P-c-Jun), phospho-IκB-α (P-IκB-α), NF-κB (p65), α-tubulin, or nucleolin (Cell Signaling Technology, Danvers, MA, USA; 1∶1000). Detection was performed with the Western blotting reagent ECL Prime (GE Healthcare, Buckinghamshire, UK). Protein levels were quantified by densitometric scanning with the Gel-Pro Analyzer (Media Cybernetics, Inc., USA) and expressed as the ratio to α-tubulin or nucleolin levels as described previously [4] (link), [14] (link).

The antibodies used were α-HA (Sigma-Aldrich H6908; WB 1 : 1,000), α-FLAG (Sigma-Aldrich F7425, WB 1 : 1,000 ChIP 5 μg), α-myc (Cell Signaling 2276 S, WB 1 : 5,000 immunofluorescence (IF) 1:150 ChIP 5 μg), α-H3K9me3 (Millipore 07–442, WB 1 : 1,000; Abcam ab8898, IF 1 : 150 ChIP 5 μg), α-H3K9ac (Cell Signaling ab1191, ChIP 5 μg), α-actin (Sigma-Aldrich A1978, WB 1 : 5,000), α-tubulin (Sigma-Aldrich T6199, WB 1 : 20,000), α-NFkB p65 (Santa Cruz Biotechnology sc-372-x, WB 1 : 1,000), α-SirT6 (AbCam ab62739, WB 1 : 1,000; ChIP 5μg), α-H3 (Cell Signaling 9715 S, WB 1 : 2,000), α-acetyl-lysine (Cell Signaling 9814 S, WB 1 : 1,000), α-suv39h1(Millipore 07–550, WB 1 : 1,000), α−SKP2 (Thermo Fisher Scientific 32–3300, WB 1 : 1,000), α-IκBα (Santa Cruz sc-371, 1 : 1,000), α-P-IκBα (Cell Signaling 9246 S, WB 1 : 1,000), and α-p100/p52 (Millipore 05-361, WB 1 : 1,000).
WBs were performed as described elsewhere. Images of original WBs included in the main figures are included in Supplementary Figure 9.