Flag tag (flag)

For expression in cultured cells, EGFP tag-fused ubiquitin, FLAG-CNPY2, FLAG-MYLIP, MYLIP-His, FLAG-UBE2D1, UBE2D1-His and deletion mutants of FLAG-ARs (full length, 1-920 a.a.; AF-2, 555-920 a.a.; AF-1, 1-673 a.a.) were inserted into the pcDNA3 vector (Invitrogen, Carlsbad, CA, USA). To produce recombinant proteins in E.coli, CNPY2, UBE2D1 and the RING domain of MYLIP (344-445 a.a.) were inserted into pET29a (+) vector (Merck Millipore, DA, Germany) or the pGEX4T1 vector (GE Healthcare, Little Chalfont, England). siRNAs for CNPY2 (CNPY2HSS115810 and 115811) and MYLIP (MYLIPHSS120911 and 120912) were purchased from Invitrogen. Data in this report was shown as results using siCNPY2 (115810) and siMYLIP (120911), which had been more efficient at knockdown or cell growth than siCNPY2 (115811) in 22Rv1 cells. A nonspecific control siRNA pool (siControl) was purchased from Dharmacon (D-001206-13-20). The following antibodies were used: AR (N-20, C-19 or 441, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CNPY2 (ab181215, Abcam, Cambridge, MA, USA), MYLIP (ab74562, Abcam), β-actin (A5441, Sigma, St. Louis, MO, USA), Ub (F-11, Santa Cruz Biotechnology), FLAG (F7425, Sigma), His (D291-3S, MBL, Nagoya, Japan), GST (B-14, Santa Cruz Biotechnology) and GFP (598, MBL).

PT2385 was obtained from Abcam, Cambridge, UK, and used at concentrations as indicated. SCD5 was subcloned by PCR from human cDNA (Agilent, Santa Clara, CA, USA), and fused by standard cloning techniques to a pcDNA6 vector encoding an N-terminal Flag-tag (Invitrogen, Carlsbad, CA, USA), and to a pLXSN vector with a Venus tag, a variant of the yellow fluorescent protein (YFP).

Groups of proteins were extracted from transfected cells. After boiling for 5 min in 5% SDS-PAGE sample loading buffer, equal amounts of protein (50 μg) samples were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked for 12 h at 4°C in PBS containing 10% skim milk. Specific antibodies against the FLAG tag (1:2,000, Invitrogen, USA), CacyBP/SIP (1:500, BOSTER, China), Caspase3 (1:300, Bioss, China), Bcl2 (1:500, BOSTER, China), Bax (1:500, BOSTER, China), and GAPDH (1:2,000, Sungene Biotech, China) were dissolved in PBS containing 1% skim milk, and then the membrane was treated with this solution. After incubation with the primary antibodies at 4°C for 12 h, the membrane was washed three times with PBST (PBS contain 0.5% Tween 20). Horseradis peroxidase-conjugated anti-rabbit/mouse IgG (1:3,000, Sungene Biotech, China) was then added. Detection was performed by incubating the membrane with Clarity Western ECL Substrate (Bio-Rad, California, USA), and then exposed the membrane in a dark room by using development and fixing baths. The film was developed artificially, and the results were analyzed using a Tanon-410 automatic gel imaging system.

The following oligos (Genewiz) were used for RNA interference:
The following primers were used for ChIP qPCR:
The following gRNA sequences were used for CRISPR/Cas9 gene editing:
Human SIRT6 was cloned into pCDNA3.1 with a FLAG tag (Invitrogen, USA); a 3 × FLAG-SIRT1 and DR-GFP plasmids were obtained from Addgene. SIRT6ΔC and ΔN were amplified with specific primers and cloned into pKH3HA (Addgene) and pGex vectors (GE Healthcare Life Sciences). The SIRT6 KR, KQ and HY mutants were obtained by converting SIRT6 lysine 33 to arginine (KR), or to glutamine (KQ) and SIRT6 133 histidine to tyrosine (HY) via site-directed mutagenesis, as described below.

Fixation and labeling of S2 cells were done as described previously (Wurm et al, 2010 ). In brief, cells were fixed using an 8% (wt/vol) formaldehyde solution, permeabilized by incubation with a 0.25% (vol/vol) Triton X-100 solution, and blocked with a 5% (wt/vol) BSA solution. Proteins of interest were labeled with antisera against the FLAG-tag (Thermo Fisher Scientific) and ATP5A (Abcam). Detection was achieved via secondary antibodies custom-labeled with Alexa Fluor 594 or STAR RED.