Anti cul4a

Manufactured by Abcam

Anti-CUL4A is a primary antibody that detects the CUL4A protein, a key component of the CUL4A-based E3 ubiquitin ligase complex. It is used for the detection and analysis of CUL4A expression in various applications, such as Western blotting and immunohistochemistry.

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Cul4A (8 citations)

5 protocols using anti cul4a

Molecular Mechanisms of CRAC Channel Regulation

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All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously^{26 (link)}. Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

Moon H., Min C., Kim G., Kim D., Kim K., Lee S.A., Moon B., Yang S., Lee J., Yang S.J., Cho S.K., Lee G., Lee C.S., Park C.S, & Park D. (2020). Crbn modulates calcium influx by regulating Orai1 during efferocytosis. Nature Communications, 11, 5489.

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Investigating ANXA10-Cul4A Interaction

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Then, 293T cells were transiently co-transfected with pBabe-Cul4A-myc-his and pCMV6-ANXA10-GFP (OriGene, Rockville, MD, USA) vectors using Lipofectamine 2000 transfection reagent (Invitrogen). Twenty-four hours after transfection, the cells were treated with 10 μg/mL of MG132 (Sigma) for 24 h, and then harvested in a NP-40 lysis buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 1% NP40), protease inhibitor, and phosphatase inhibitor cocktail (Roche, Lewes, UK, USA). Immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore) according to the manufacturer’s protocols. Anti-GFP (OriGene) and Anti-Cul4A (Abcam) antibodies were used for immunoprecipitation.

Hung M.S., Chen Y.C., Lin P.Y., Li Y.C., Hsu C.C., Lung J.H., You L., Xu Z., Mao J.H., Jablons D.M, & Yang C.T. (2019). Cul4A Modulates Invasion and Metastasis of Lung Cancer through Regulation of ANXA10. Cancers, 11(5), 618.

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Western Blot Analysis of Osteogenic and Neurogenic Markers

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Cells and tissue samples were lysed in RIPA buffer (Cat. #89900; Thermo Fisher Scientific) supplemented with 1× protease inhibitor cocktail (Cat. #11697498001; Roche, USA). The cell lysates were resolved by 12% SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes and probed with primary and secondary antibodies, respectively. The following primary antibodies were used: anti-CtBP1 (Cat. #612042; BD Biosciences, USA), anti-CtBP2 (Cat. #612044; BD Biosciences), anti-OSC (Cat. #ab93876; Abcam, USA), anti-ALPL (Cat. #ab116592; Abcam), anti-COL1A1 (Cat. #PA1-26204; Thermo Fisher Scientific), anti-IBSP (Cat. #PA5-41327; Thermo Fisher Scientific), anti-SPP1 (Cat. #HPA027541; Sigma-Aldrich), anti-MMP13 (Cat. #ab39012; Abcam), anti-CUL4A (Cat. #ab92554; Abcam), anti-MAP2 (Cat. #ab5392; Abcam), anti-DCX (Cat. #AV41333; Sigma-Aldrich), anti-NSE (Cat. #SAB4300698; Sigma-Aldrich), and anti-GAPDH (Cat. #ab8254; Abcam). The secondary antibodies included anti-mouse IgG (Cat. #ab6728; Abcam) and anti-rabbit IgG (Cat. #ab6721; Abcam).

Chen X., Zhang W., Zhang Q., Song T., Yu Z., Li Z., Duan N, & Dang X. (2020). NSM00158 Specifically Disrupts the CtBP2-p300 Interaction to Reverse CtBP2-Mediated Transrepression and Prevent the Occurrence of Nonunion. Molecules and Cells, 43(6), 517-529.

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Confocal Microscopy-Based Colocalization Analysis

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U2OS cells were labeled with EdU for 30 min, rinsed in phosphate buffered saline (PBS) pH 7.4 and then pre-extracted in PBS-T buffer (0.2% Triton X-100 in 1× PBS, phenylmethylsulphonylfluoride [PMSF], protease inhibitor cocktail [Sigma, P8340] and phosphatase inhibitor cocktail [Roche, P4906845001]) for 5 min on ice, followed by 2.0% paraformaldehyde (PFA). Primary antibody staining was performed as follows: anti-FLAG (1:1000, Sigma, F1804), anti-CUL4A (1:200, Abcam, ab92554), anti-γH2AX (1:500, Millipore, 05-636), anti-pRPA (1:500, Bethyl labs, A300-245A) and anti-PCNA (1:200, Santa Cruz, sc-7907). Secondary antibody staining was performed as follows: Alexa 488 conjugated anti-mouse IgG and Alexa 555 conjugated anti-rabbit IgG (1:500, Thermo Fisher Scientific, A11029 and A21428). EdU was detected by Click-iT assay kit according to the manufacturer’s protocol. A Zeiss LSM710 confocal microscope was used and Pearson’s correlation coefficient (R, or Rcoloc) that is the covariance of the two variables divided by the product of their standard deviations was used for colocalization analysis and calculated using the colocalization Plugin of the FIJI-ImageJ software (https://imagej.nih.gov/ij/index.html). Detailed results of the colocalization analyses are presented in Supplementary Dataset.

Jang S.M., Zhang Y., Utani K., Fu H., Redon C.E., Marks A.B., Smith O.K., Redmond C.J., Baris A.M., Tulchinsky D.A, & Aladjem M.I. (2018). The replication initiation determinant protein (RepID) modulates replication by recruiting CUL4 to chromatin. Nature Communications, 9, 2782.

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Chromatin Immunoprecipitation of K562 Cells

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Chromatin immunoprecipitation (ChIP) analyses were performed on 1% formaldehyde-fixed proliferating or differentiating K562 cells using a Pierce^™ magnetic ChIP assay kit (Thermofisher Scientific, 26157). Antibodies used in this study included normal rabbit IgG (Cell Signaling, 2729, 1:500), anti-CUL4A (Abcam, ab92554, 1:100), anti-JARID1A (Abcam, ab78322, 1:100), anti-H3K4me3 (Millipore, 05–1339, 1:100) and anti-H3K9me3 (Millipore, 05–1250, 1:100). Immunoprecipitated DNA and control input DNA was analyzed by quantitative real-time PCR using the human DAB2 promoter-specific primers; forward:5’-CTC GCG GAG CTC AGG GGA G-3,’ reverse:5’-GGT AAC TCC CCC TCA ACG TG-3. ‘

Jo J.H., Ok S.M., Kim D.K., Kim Y.M., Park J.U., Jung D.H., Kim H.J., Seong H.A., Cho H.J., Nah J., Kim S., Fu H., Redon C.E., Aladjem M.I, & Jang S.M. (2023). RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter. Research Square.

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