Anti cullin 1

The tissue embedded in paraffin were fixed and dewaxed, followed by endogenous peroxidase blocking with 3% hydrogen peroxide solution at room temperature for 30 min. Tissues were steamed for 30 min in citrate buffer to repair antigens and incubated with 5% BSA solution for 30 min to block non‐specific antigens. The tissues were then incubated with antibodies: anti‐NEDD8 (1:200; CST), and anti‐cullin 1 (1:200; Abcam) at 4°C for overnight, and with the secondary antibody at 37°C for 30 min. Subsequently, tissue color rendering by DAB (Biorad) was used, resin sealing applied, and pathological section scanner used to obtain pictures.

FKB with 99% purity was isolated from kava extracts by LKT Laboratories, Inc. (St. Paul, MN). Bortezomib, MG-132 and MLN4924 were obtained from Cayman Chemical Inc. (Ann Arbor, MI). Antibodies against Ubc12, ubiquitin, and β-tubulin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Skp2 antibodies were purchased from Invitrogen (Grand Island, NY). Anti-Myc-tag and Anti-cleaved-PARP antibodies were from Cell Signaling (Boston, MA). Anti-Cullin-1 and anti-NEDD8 antibodies were from Abcam (Cambridge, MA). Anti-p27/Kip1 and p21/WAF1 antibodies were from BD Biosciences (Billerica, MA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma. The Reverse Transcription System kit and was from Promega (Mandison, WI). A quantitative reverse transcription polymerase chain reaction (RT-PCR) kit was from Bio-Rad (Hercules, CA). Ubiquitylation Assay Kit and 20S Proteasome Assay Kit were from Cayman and Abcam, respectively.

RIPA lysate (Pierce) was added to the tissues/cells to obtain the protein that was mixed with the loading buffer (Life Technologies) at a ratio of 3:1, and boiled at 100°C for 8‐10 min. Protein samples were added to the gel well and the target protein was separated by electrophoresis. The protein was transferred to a PVDF membrane (Millipore), and non‐specific antigens blocked with 5% skim milk. The membranes were incubated with corresponding primary antibodies: anti‐CD9 (1:500; Proteintech), anti‐CD63 (1:500; Proteintech), anti‐CD81 (1:500; Proteintech), anti‐NEDD8 (1:500; CST), anti‐cullin 1 (1:500; Abcam), anti‐IκB (1:500; CST), anti‐NF‐κB (1:500; Abclonal), anti‐P‐NF‐κB (1:500; Abclonal), anti‐PCNA (1:500; Abcam), and anti‐β‐actin (1:500; SAB) overnight at 4°C. After washing with 1× TBS/T buffer, the membranes were incubated with the secondary antibody for 30 min at 37°C. Pictures were taken with a chemical gel imaging system (GE).

Antibodies used in this work: Anti-Akt (CST, #9272, 1:1,000), anti-pThr308-Akt (CST, #9275, 1:1,000), anti-pSer473-Akt (CST, # 4060, D9E, 1:1,000), anti-p70 S6K (CST, #9202, 1:1,000), anti-pThr389-p70 S6K (CST, no. 9209, 1:1,000), anti-pSer235/236-S6 (CST, #4858, 1:1,000), anti-S6 (CST, #2317, 1:1,000), anti-4E-BP1 (CST, #9452, 1:1,000), anti-pSer65-4E-BP1 (CST, #9451, 1:1,000), anti-PTEN (CST, #9559, 1:1,000), anti-pSer2448-mTOR (CST, # 5536, 1:500), anti-mTOR (CST, #2972, 1:500), anti-UBE1a (CST, # 4890, 1:1,000) and anti-pAMPK (CST, #2535, 1:1,000) were purchased from Cell Signaling Technology. Anti-GAPDH (Santa Cruz, sc-293335, 1:1,000), Anti-UBA3 (Santa Cruz, sc-377212, 1:200), anti-NAE1 (Santa Cruz, sc-390002, 1:200) and anti-NEDP1 (Santa Cruz, sc-271498, 1:100) were from Santa Cruz Biotechnologies. Anti-UBE2D3 (Abcam, ab176568, 1:1,000), anti-Cullin1 (Abcam, ab75817, 1:1,000) and anti-SAE1 (Abcam, ab185552, 1:1,000) were purchased from Abcam. Anti-Nedd8-K402-PTEN antibody was from PTM Biolabs, Inc. The NAE inhibitor MLN4924 (HY-70062), 2-Deoxy-D-glucose (a glucose analog and a competitive inhibitor of glucose metabolism) (2-DG, HY-13966), 5-Azacytidine (Azacitidine; 5-AzaC; Ladakamycin) (HY-10586) were purchased from MCE.

Cells obtained from culture were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 30 min. Triton X‐100 (0.1%) was added, gently mixed for 30 min, and non‐specific antigens blocked by the addition of 5% BSA solution. The cells were incubated with the antibodies: anti‐NEDD8 (1:200; CST), and anti‐cullin 1 (1:200; Abcam) overnight at 4°C, then incubated with fluorescent secondary antibody at 37°C for 60 min. They were finally incubated with honchest (Biorad) for 10 min at room temperature, and anti‐quenching agent, after which pictures were taken by scanning with a confocal laser microscope (Nikon). The light was avoided in the entire process.