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Cul-5 is a laboratory equipment product from Santa Cruz Biotechnology. It is a component of the cullin-RING E3 ubiquitin ligase complex, which plays a role in the ubiquitination and proteasomal degradation of target proteins.

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Lab products found in correlation

Total fak, by BD (2 mentions) Anti flag clone m2, by Merck Group (2 mentions) Anti α tubulin, by Santa Cruz Biotechnology (2 mentions) Fak s910, by Thermo Fisher Scientific (2 mentions) Anti rab40c, by Santa Cruz Biotechnology (2 mentions) Mouse ankrd28, by Santa Cruz Biotechnology (2 mentions) Mob1 e1n9d, by Cell Signaling Technology (2 mentions) P mob1 d2f10, by Cell Signaling Technology (2 mentions) Anti saps1 2 3, by Fortis Life Sciences (2 mentions) Ankrd28 52, by Fortis Life Sciences (2 mentions) Anti ha, by Santa Cruz Biotechnology (2 mentions) β actin, by Merck Group (2 mentions) Cul4a, by Cell Signaling Technology (2 mentions) Cul4b, by Proteintech (2 mentions) Ube2m, by Santa Cruz Biotechnology (2 mentions) Flag bead, by Merck Group (1 mentions) Normal igg ac, by Santa Cruz Biotechnology (1 mentions) Nedd4 1, by Santa Cruz Biotechnology (1 mentions) Nedd4 1, by Cell Signaling Technology (1 mentions) Protein a g bead, by Santa Cruz Biotechnology (1 mentions)

6 protocols using cul 5

1

Antibody Verification and Characterization

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The following antibodies were used in this study: anti-FLAG (clone M2, WB 1:1,000; Sigma-Aldrich), anti-GAPDH (WB 1:5,000; UBPBio), FAK S910 (WB 1:1,000, 44-596G; Invitrogen), total FAK (WB 1:1,000, 610087; BD Biosciences), and paxillin (IF 1:500; Transduction Labs). Anti-HA (WB 1:500, IP 2 μg/1 mg cell lysate, SC F-7), anti–α-tubulin (WB 1:5,000, 23948), anti-Rab40c (WB 1:500, H-8 sc514826), cul-5 (WB 1:500, H-300), and mouse ANKRD28 (WB 1:500) were purchased from Santa Cruz Biotechnology. MOB1(E1N9D) and p-MOB1(D2F10) were purchased from Cell Signaling Technology. Rabbit anti-SAPS1/2/3, ANKRD28/52, and PP6c were purchased from Bethyl Laboratories. Specificity of anti-Rab40c antibody was confirmed by immunoblotting lysates derived from cells expressing FLAG-Rab40a, FLAG-Rab40b, or FLAG-Rab40c constructs (Fig S2A).
Han K.J., Mikalayeva V., Gerber S.A., Kettenbach A.N., Skeberdis V.A, & Prekeris R. (2022). Rab40c regulates focal adhesions and PP6 activity by controlling ANKRD28 ubiquitylation. Life Science Alliance, 5(9), e202101346.
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2

Immunoprecipitation of Endogenous and Exogenous Proteins

Cited 1 time
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To immunoprecipitate endogenous proteins, whole cell extracts were pre-cleared with normal IgG-AC (Santa Cruz) followed by overnight incubation at 4°C with antibody against NEDD4-1 (Santa Cruz; #sc-25508) and SAG [11 (link)]. To immunoprecipitate exogenously expressed FLAG-tagged or HA-tagged proteins, the pre-cleared cell lysates were incubated with FLAG beads (Sigma) or with HA antibody (Roche) for 3 hrs followed by incubation with protein A&G beads (Santa Cruz) at 4°C overnight with rotation. The beads were washed three times with lysis buffer, and the immunoprecipitation complexes were subjected to SDS-PAGE.
Whole-cell lysates were prepared and subjected to immunoblotting analysis using antibodies against SAG [11 (link)], ROC1 [42 (link)], HA (Roche), FLAG (Sigma), CUL-5 (Santa Cruz), NEDD4-1 (Cell Signaling; #3607), Parp (Cell signal), Cleaved-caspase-3 (Cell Signal), and β-actin (Sigma)
Zhou W., Xu J., Zhao Y, & Sun Y. (2014). SAG/RBX2 is a novel substrate of NEDD4-1 E3 ubiquitin ligase and mediates NEDD4-1 induced chemosensitization. Oncotarget, 5(16), 6746-6755.
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3

Western Blot Analysis of HIV-1 Vif Interactors

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Proteins were separated by SDS-PAGE, followed by transfer to nitrocellulose membrane (Bio-Rad). After blocking with PBS-buffered saline-Tween 20 containing 5% BSA for 20 min at room temperature, membranes were incubated with a specific antibody overnight at 4°C. After three washes with PBS-buffered saline-Tween 20, the membranes were stained with an alkaline phosphatase-conjugated secondary antibody (1:3,000, Jackson Immunoresearch) for 2 h at room temperature. After three washes with PBS-buffered saline-Tween 20, the membranes were incubated in development buffer containing 5-bromo-4-chloro-3′-indolylphosphate (BCIP) and nitro-blue tetrazolium (NBT) substrate (Sigma). The antibodies used in this study were specific for: Vif (the AIDS Research Reagents Program, catalog 2221), Cul5 (Santa Cruz Biotechnology Inc., catalog sc-13014), CBF-β (Abcam, catalog ab11921), Elo B (Santa Cruz Biotechnology, Inc, catalog sc-11447), Elo C (BD Transduction Laboratories, catalog 610760), HA (Invitrogen, catalog 715500), and V5 (Invitrogen, catalog R96025).
Evans S.L., Schön A., Gao Q., Han X., Zhou X., Freire E, & Yu X.F. (2014). HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3. Retrovirology, 11, 4.
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4

Western Blot Analysis of Cullin Proteins

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PBS-washed cell pellets were lysed in 50mM Tris pH 7.9, 8M Urea and 1% CHAPS and incubated with shaking at 4ºC for at least 30min. 20μg of supernatants were run and transferred for detection. Antibodies used: CUL1 (1:1000, Santa Cruz Biotechnology, sc-1276), CUL2 (1:500, Sigma-Aldrich, SAB2501565-100), CUL3 (1:1000, Cell Signaling Technology, 2759), CUL4A (1:1000, Cell Signaling Technology, 2699S), CUL4B (1:1000, Proteintech, 12916-1-AP), CUL5 (1:1000, Santa Cruz Biotechnology, sc-373822), UBE2M (1:1000, Santa Cruz Biotechnology, sc-390064), DDB1 (1:1000, Cell Signaling Technology, 5428S), cyclin K (1:5000, Bethyl, A301-939A), CDK12 (1:1000, Cell Signaling Technology, 11973S), CDK13 (1:1000, Bethyl, A301-458A), RBM39 (1:500, Santa Cruz Biotechnology sc-376531), V5 (1:1000 Cell Signaling Technology, 13202), Ubiquityl-Histone H2A (K119) (1:1000, Cell Signaling, 8240-20), MLH1 (1:1000, Cell Signaling Technology, 3515T). ACTIN (1:10000, Sigma-Aldrich, A5441), VINCULIN (1:1000, Santa Cruz Biotechnology, sc-25336). Secondary antibodies anti-mouse/rabbit/goat (1:10000, Jackson ImmunoResearch 115-035-003, 111-035-003 and 705-035-003) and anti-rabbit Alexa Fluor 488 (1:1000, Invitrogen, A21441).
Mayor-Ruiz C., Bauer S., Brand M., Kozicka Z., Siklos M., Imrichova H., Kaltheuner I.H., Hahn E., Seiler K., Koren A., Petzold G., Fellner M., Bock C., Müller A.C., Zuber J., Geyer M., Thomä N.H., Kubicek S, & Winter G.E. (2020). Rational discovery of molecular glue degraders via scalable chemical profiling. Nature chemical biology, 16(11), 1199-1207.
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5

Analysis of Cullin-RING Ubiquitin Ligases

Cited 1 time
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Cells after indicated treatment were collected, lysed and examined by standard western blot procedures. SAG monoclonal Ab was raised against the RING domain (AA44-113) [42] (link). The other antibodies were purchased from a variety of vendors as follows: CUL1CTD (Proteintech, 12895-1-AP), CUL1 (Santa Cruz, SC-11384), CUL5CTD (Sigma-Aldrich, AV35127), CUL5 (Santa Cruz, sc-373822), CUL3 (Cell signaling, 2759S), CUL2 (abcam, ab166917), CUL4A (Cell signaling, 2699S), CUL4B (Proteintech, 12916-1-AP), RBX1 (Cell signaling, 11922S), NEDD8 (abcam, ab81264), UBA3 (abcam, ab124728), NAE1 (Cell signaling, 14321S), UBE2F (Santa Cruz, sc-398668), UBE2M (Santa Cruz, sc-390064), p21 (Cell signaling, 2947S), p27 (Cell signaling, 2552S), MCL1 (Cell signaling, 5453S), NRF2 (Santa Cruz, sc-722), CDT1 (Santa Cruz, sc-28262), NOXA (EMD Millipore, OP180), and β-Actin (Sigma-Aldrich, A5441).
Yu Q., Hu Z., Shen Y., Jiang Y., Pan P., Hou T., Pan Z.Q., Huang J, & Sun Y. (2020). Gossypol inhibits cullin neddylation by targeting SAG-CUL5 and RBX1-CUL1 complexes. Neoplasia (New York, N.Y.), 22(4), 179-191.
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6

Antibody Characterization and Validation

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The following antibodies were used in this study: anti-FLAG (clone M2, WB 1:1,000, Sigma), anti-GAPDH (WB 1:5,000, UBPBio), FAK S910 (WB 1:1,000, 44-596G, Invitrogen), total FAK (WB 1:1,000, 610087, BD Biosciences), paxillin (IF 1:500 Transduction labs). Anti-HA (WB 1:500, IP 2 µg/1 mg cell lysate, SC F-7), anti-α-tubulin (WB 1:5,000, 23948), anti-Rab40c (WB 1:500, H-8 sc514826), cul-5 (WB 1:500, H-300), and mouse ANKRD28 (WB 1:500) were purchased from Santa Cruz Biotechnology. MOB1(E1N9D) and p-MOB1(D2F10) were purchased from cell signaling technology. Rabbit anti-SAPS1/2/3, ANKRD28/52, PP6c were purchased from Bethyl Laboratories.
Han K., & Prekeris R. (2021). Rab40c Regulates Focal Adhesions and Protein Phosphatase 6 Activity by Controlling ANKRD28 Ubiquitylation and Degradation.
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