Cullin1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Cullin1 is a protein that plays a critical role in the regulation of the cell cycle and the ubiquitin-proteasome system. It serves as a scaffold for the assembly of E3 ubiquitin ligase complexes, which are responsible for the targeted degradation of specific substrate proteins.

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6 protocols using cullin1

Immunoblotting Analysis of Cellular Signaling

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Cell lysates were prepared for immunoblotting, using antibodies against cullin1 (Santa Cruz, Dallas, Texas), phospho-H2AX at Ser139 (γH2AX), ATG7, cleaved caspase 3, cleaved PARP, Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit, Pro-Survival Bcl-2 Family Antibody Sampler Kit, IAP Family Antibody Sampler Kit, ORC1, CDT1 (Cell Signaling, Boston, MA), tubulin (Likun Trade Co., China), NOXA (Millipore, Billerica, MA) and LC3 (Sigma, St. Louis, MO).

Chen P., Hu T., Liang Y., Jiang Y., Pan Y., Li C., Zhang P., Wei D., Li P., Jeong L.S., Chu Y., Qi H., Yang M., Hoffman R.M., Dong Z, & Jia L. (2015). Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer. Oncotarget, 6(11), 9002-9017.

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Western Blot Analysis of KSHV Proteins

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Western blot analysis was carried out as described previously (47 (link)). Briefly, protein samples were resolved on an 8% to 12% polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) where the target protein was probed with a specific antibody. All Western blot analyses done in the study were performed using at least two independent sets of cell lysates, and similar results were obtained across all experiments. The anti-ORF50 antibody was generated as described previously (48 (link)). Antibodies to FLAG (A8592; Sigma), K8 (sc-57889; Santa Cruz), ORF45 (sc-53883; Santa Cruz), K8.1 (sc-65446; Santa Cruz), LANA (13-210-100; Advanced Biotechnologies), RBP-Jκ (sc-271128; Santa Cruz), KAP1 (ab-22553; Abcam), Nrf2 (sc-722; Santa Cruz), HIF-1α (610958; BD Biosciences), Fos (sc-52; Santa Cruz), Jun (sc-1694; Santa Cruz), phospho-Jun (13-2100; Advanced Bioteck, Inc.), SP1 (CS200631; Millipore), hemagglutinin (901501; BioLegend), NEDD8 (PA5-17476; Thermo Fisher), cullin 1 (sc-11384; Santa Cruz), PARP (9532; Cell Signaling), cleaved caspase-3 (9664; Cell Signaling), and actin (sc-47778; Santa Cruz) were purchased commercially.

Chang P.J., Chen L.W., Chen L.Y., Hung C.H., Shih Y.J, & Wang S.S. (2017). Effects of the NEDD8-Activating Enzyme Inhibitor MLN4924 on Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus. Journal of Virology, 91(19), e00505-17.

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Western Blot Analysis of Cellular Proteins

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Cell lysates were quantified with the Bio-Rad protein assay kit II (Bio-Rad Laboratories, 500-0002EDU). The boiled lysates were resolved by SDS-PAGE, transferred to PVDF membranes and probed with the primary antibodies for various time, The membranes were washed with TBS-T (0.01 M TRIS-HCl Buffer, 8.8 g/L NaCl, 0.1% Tween-20), then incubated with suitable HRP-conjugated second antibodies (Dako, P016102 and P021702). Protein bands were visualized using standard chemical luminescence methodology.
The sources of primary antibodies were as follows: SAG monoclonal Ab was raised against the RING domain (AA44-113)50 (link), cullin1 (Santa Cruz Biotechnology, CA, USA), RBX1, caspase-3, PARP, ORC1, CDT1, p16, p21, p27, BIM, cyclin B1,E-Cadherin, N-Cadherin, Vimentin, GAPDH, phospho-mTOR (Ser2448), phospho-Akt (Ser473), phospho-CHK1 (Ser345), phospho-CHK2 (Thr68), phospho-Histone H3 (Ser10), phospho-p70 S6 (Thr389), phospho-4E-BP1 (Thr70), phosphor-IkBα (Ser 32), IkBα and DEPTOR (Cell Signaling Technology, Denver, CO, USA), phospho-H2A.x (Ser139) (Millipore, Bedford, MA, USA), NEDD8, NOXA, PHLPP1 (Abcam, Cambridge, MA), SQSTM1/p62, LC3 (Sigma, St. Louis, MO), phospho-ATM (Ser1981) (Rockland, Limerick, PA), HIF-1α (Novus Biologicals, Littleton, CO, USA).

Lan H., Tang Z., Jin H, & Sun Y. (2016). Neddylation inhibitor MLN4924 suppresses growth and migration of human gastric cancer cells. Scientific Reports, 6, 24218.

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Immunoblotting Analysis of Cell Lysates

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Cell lysates after various treatments were prepared and subjected to IB analysis as described (Gu et al., 2007 (link)). The sources of primary antibodies were as follows: Cullin-1 (Santa Cruz, CA), phospho-AKT-Ser473, phosphor-AKT-Thr308, pan-AKT, AKT1, AKT2, AKT3, Aurora A, VHL, Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11), PDK1, phospho-PDK1-Ser²⁴¹ and Rictor (53A2) (Cell Signaling, Denver, CO), γ-tubulin, α-tubulin Clone AA13, acetylated tubulin Clone 6-11B-1, β-actin (Sigma, St. Louis, MO), CP110, NDE1, Cep97, Arl13B (Proteintech, Wuhan, P.R.C). UBA3 (Abcam, Shanghai, P.R.C). EGFR and phospho-EGFR-Tyr845 were gifts from Dr. Wanhong Xu,

Mao H., Tang Z., Li H., Sun B., Tan M., Fan S., Zhu Y, & Sun Y. (2019). Neddylation inhibitor MLN4924 suppresses cilia formation by modulating AKT1. Protein & Cell, 10(10), 726-744.

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Immunoblotting Analysis of Cell Signaling

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Immunoblotting was performed as previously described [14 (link),16 (link)]. Cell lysates were prepared for immunoblotting, using antibodies against NEDD8, NAE1, UBA3, UBC12, WEE1, cullin-1(Santa Cruz Biotechnology), ORC1, p27, cleaved Caspase 3, cleaved PARP, CDT1, H2AX, p-H2AX, CHK1, p-CHK1, GAPDH, p-Histone H3 (Cell Signaling), IκB-α, p-IκB-α, p21 (Epitomics, Inc.).

Gao Q., Yu G.Y., Shi J.Y., Li L.H., Zhang W.J., Wang Z.C., Yang L.X., Duan M., Zhao H., Wang X.Y., Zhou J., Qiu S.J., Jeong L.S., Jia L.J, & Fan J. (2014). Neddylation pathway is up-regulated in human intrahepatic cholangiocarcinoma and serves as a potential therapeutic target. Oncotarget, 5(17), 7820-7832.

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Immunoblotting Analysis of Cell Signaling

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Cell lysates were prepared for immunoblotting using antibodies against Wee1, cullin-1, NF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ORC1, P27, cleaved Caspase 3, cleaved PARP, CDT1, H2AX, p-H2AX, CHK1, p-CHK1, Bcl-2,Bcl-xL, Mcl-1, c-IAP1, c-IAP2, Survivin, XIAP, BAX, Bik, Bim, BID, Bak, ROC1 (Cell Signaling Technology, CST, Danvers, MA, USA), IκB-α, p-IκB-α, P21, RhoA, Cdc42 (Epitomics, Burlingame, CA, USA), NOXA (CALBIOCHEM, San Diego, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma).

Yao W.T., Wu J.F., Yu G.Y., Wang R., Wang K., Li L.H., Chen P., Jiang Y.N., Cheng H., Lee H.W., Yu J., Qi H., Yu X.J., Wang P., Chu Y.W., Yang M., Hua Z.C., Ying H.Q., Hoffman R.M., Jeong L.S, & Jia L.J. (2014). Suppression of tumor angiogenesis by targeting the protein neddylation pathway. Cell Death & Disease, 5(2), e1059-.

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