pcdna3-ha2-cul4a  (Addgene inc)

Journal: iScience

Article Title: Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

doi: 10.1016/j.isci.2024.108933

Figure Lengend Snippet: Expression of CRL4 during neuronal development (A and B) Representative immunoblots (A) and relative levels normalized by Actb (B) of CRL4 proteins in cultured cerebral neurons (n = 3; ∗p < 0.05, DIV5 versus DIV10 by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (C) Representative immunoblots of Cul4a and Cul4b in DIV5 neurons treated with or without MLN4924 or CSN5i-3 for 24 h. Arrows indicate the neddylated forms of Cul4a and Cul4b. (D and E) The left graphs represent the portions of neddylated and native forms of Cul4a (D) and Cul4b (E) at DIV5 and DIV10. The graphs on the right show the ratio of neddylated versus native form (n = 3; ∗p < 0.05, DIV5 versus DIV10 by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (F and G) Representative images of the immunocytochemistry of Cul4a (F) or Cul4b (G) with Ddb1 in DIV5 and DIV10 neurons (Scale bar, 10 μm). The graphs on the right show the intensity of immunoreactivities of Cul4a or Cul4b with Ddb1 and DAPI in dotted lines (1–2). (H and I) Representative immunoblots (H) and relative levels (I) of Cul4a, Cul4b, and Ddb1 in total, nuclear (nuc) and cytosolic (cyto) fractions of in vitro cultured neurons (∗p < 0.05, DIV5 versus DIV10 by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (J and K) Representative images for immunohistochemistry of Cul4a (J) or Cul4b (K) with Ddb1 in cortical plate of embryonic day 18 (E18) and cortical layer V of adult rat brains (Scale bar, 50 μm). (L) Graphic summary of cytosolic and nuclear expression of Cul4a, Cul4b, and Ddb1. The expression level is visualized by red, green, and blue intensity, respectively.

Article Snippet: For genetic perturbation of CRL4, pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909).

Techniques: Expressing, Western Blot, Cell Culture, Two Tailed Test, Immunocytochemistry, In Vitro, Immunohistochemistry

Journal: iScience

Article Title: Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

doi: 10.1016/j.isci.2024.108933

Figure Lengend Snippet: Expression and neddylation of Cul4a and Cul4b are controlled by neuronal activity (A) Schematic diagram of experiment (Glu, glutamate; AP5, D-AP5; LY, LY341495; MEM, memantine, NMDAR, NMDA receptor; AMPAR, AMPA receptor; KAR, Kainate receptor; mGluRs, metabotropic glutamate receptors). (B) Representative immunoblots of CRL4 proteins in DIV10 neurons stimulated by 25 μM Glu for 10 min with or without pre- and post-treatments of antagonists of glutamate receptor (50 μM AP5, 10 μM NBQX, or 10 μM LY). Arrows indicate the neddylated forms of Cul4a and Cul4b. (C‒E) The relative level of Ddb1 (C) and total Cul4a (D) is normalized by Actb. (E) Relative levels of neddylated, native forms and the neddylation ratio (neddylated versus native) of Cul4a (n = 3, ∗p < 0.05 to the no-treatment group, †p < 0.05 to the glutamate-treatment group in the same treatment time by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (F) Representative images (upper panels) and relative intensities (below panels) of the immunocytochemistry of Cul4a and Ddb1 in neurons stimulated by glutamate with or without AP5. (G-H) The relative level of total Cul4b (G) is normalized by Actb. (H) Relative levels of neddylated, native forms and the neddylation ratio (neddylated versus native) of Cul4b (n = 3, ∗p < 0.05 to the no-treatment group, †p < 0.05 to the glutamate-treatment group in the same treatment time by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (I) Representative immunoblot of CRL4 proteins under 100 μM NMDA stimulation with or without 10 μM MEM. Arrows indicate the neddylated forms of Cul4a and Cul4b. (J‒L) The relative level of Ddb1 (J) and total Cul4a (K) is normalized by Gapdh. (L) Relative levels of neddylated, native forms and the neddylation ratio (neddylated versus native) of Cul4a (n = 3, ∗p < 0.05 to the no-treatment group, †p < 0.05 to NMDA-treatment group in the same treatment time by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (M) Representative images of the immunocytochemistry of Cul4a and Ddb1 in neurons stimulated by NMDA with or without MEM. The below panels indicate relative intensities in the dotted line (1-2). (N-O) The relative level of total Cul4b (N) is normalized by Gapdh. (O) Relative levels of neddylated, native forms and the neddylation ratio (neddylated versus native) of Cul4b (n = 3, ∗p < 0.05 to the no-treatment group, †p < 0.05 to NMDA-treatment group in the same treatment time by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (P) Summary of change of expression, activity, and localization of Cul4a and Cul4b under neuronal activation.

Article Snippet: For genetic perturbation of CRL4, pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909).

Techniques: Expressing, Activity Assay, Western Blot, Two Tailed Test, Immunocytochemistry, Activation Assay

Journal: iScience

Article Title: Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

doi: 10.1016/j.isci.2024.108933

Figure Lengend Snippet: CRL4 interacts with cytoskeletal proteins in developing neurons (A) Experimental strategy for identification of Cul4a- and Cul4b-binding proteins. (B) Cul4a- and Cul4b-immunoprecipitates from lysates of DIV5 and DIV10 neurons. Proteins loaded onto polyacrylamide gel were stained by Coomassie blue. HC and LC indicate heavy and light chains of immunoprecipitated anti -Cul4a- and anti -Cul4b antibodies, respectively. (C and D) Gene ontology and Reactome analyses of Cul4a- and Cul4b-binding proteins. Top GO:BP (biological process), GO:CC (cellular component), GO:MF (molecular function), and REAC (reactome) having -Log(p value) was over 2 of DIV5 (C) and DIV10 (D) neurons. (E) Selected list of proteins involved in cytoskeletal regulation and/or neuronal morphogenesis in the proteome of DIV10 neuron. Proteins within the pink box are related to cytoskeleton regulation, and those in the blue box are related to neuronal morphogenesis.

Article Snippet: For genetic perturbation of CRL4, pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909).

Techniques: Binding Assay, Staining, Immunoprecipitation

Journal: iScience

Article Title: Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

doi: 10.1016/j.isci.2024.108933

Figure Lengend Snippet: Genetic depletion of CRL4 induces neurite extension and branching in early neuronal development (A) Representative images of the morphology of DIV4 neurons expressing the sgLacZ, sgCul4a, or sgCul4b (Scale bar, 30 μm). (B) Sholl analyses of axon and dendrite. The number of intersections between neurites and concentric shells having 25 or 5 μm intervals (n = 100, ∗p < 0.05 to control group by Mann-Whitney rank-sum test; Data are represented as mean ± SEM.). (C‒F) Average (left panels) and cumulative frequency (right panels) of length (C) and branch number (E) of total neurite, and length (D) and branch number (F) of axon/dendrite (n = 100, ∗p < 0.05 to control group by Mann-Whitney rank-sum test; Data are represented as mean ± SEM.).

Article Snippet: For genetic perturbation of CRL4, pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909).

Techniques: Expressing, Control, MANN-WHITNEY

Journal: iScience

Article Title: Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

doi: 10.1016/j.isci.2024.108933

Figure Lengend Snippet: Inhibition of cytosolic CRL4 enhances neurite morphogenesis in developing neurons (A) Structural domains of DN-Cul4 and DN-Cul4b. (B and C) Immunofluorescence images of DN-Cul4a-Flag (B) and DN-Cul4b-Flag (C) in transfected DIV4 neurons (Scale bar, 20 μm). (D) Representative images of the morphology of DIV4 neurons expressing the control vector, DN-Cul4a or DN-Cul4b (Scale bar, 30 μm). (E) Sholl analyses of axon and dendrite. The number of intersections between neurites and concentric shells having 20 or 5 μm intervals (n = 50, ∗p < 0.05 to control group by one-way ANOVA on ranks using Dunnett’s Method; Data are represented as mean ± SEM.). (F‒I) Average (left panels) and cumulative frequency (right panels) of length (F) and branch number (G) of total neurite, and length (H) and branch number (I) of axon/dendrite (n = 50, ∗p < 0.05 to control group by one-way ANOVA on ranks using Dunnett’s Method; Data are represented as mean ± SEM.).

Article Snippet: For genetic perturbation of CRL4, pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909).

Techniques: Inhibition, Immunofluorescence, Transfection, Expressing, Control, Plasmid Preparation

Journal: iScience

Article Title: Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

doi: 10.1016/j.isci.2024.108933

Figure Lengend Snippet: Overexpression of Cul4a or Cul4b abolishes NMDA-induced axonal extension (A) Immunoblot of CRL4 proteins under the 100 μM NMDA treatment for 24 h in DIV5 neurons. (B and C) Quantification of relative protein level and neddylation ratio of Cul4a (B) and Cul4b (C) normalized by Actb level (n = 3, ∗p < 0.05 to no-NMDA group by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (D) Representative images of the morphology of DIV4 neurons overexpressing Mock, Cul4a+Ddb1, or Cul4b+Ddb1 with or without 1 μM NMDA for 48 h (Scale bars, 50 μm). (E) Sholl analyses of axon and dendrite of Mock-expressing neurons with or without NMDA. Average of intersections between neurites and concentric shells having 20 or 5 μm interval (n = 50, ∗p < 0.05 to no-NMDA group by Mann-Whitney rank-sum test; Data are represented as mean ± SEM.). (F) Sholl analyses of axon and dendrite of Mock with no-NMDA, Cul4a+Ddb1 with NMDA, or Cul4b+Ddb1 with NMDA. (G‒J) Average (left panels) and cumulative frequency (right panels) of length (G) and branch number (H) of total neurite, and length (I) and branch number (J) of axon/dendrite. (n = 50, ∗p < 0.05 to no-NMDA group by Mann-Whitney rank-sum test; †p < 0.05 to Mock+NMDA group by one-way ANOVA on ranks using Dunnett’s Method; Data are represented as mean ± SEM.).

Article Snippet: For genetic perturbation of CRL4, pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909).

Techniques: Over Expression, Western Blot, Two Tailed Test, Expressing, MANN-WHITNEY

Journal: iScience

Article Title: Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment

doi: 10.1016/j.isci.2024.108933

Figure Lengend Snippet: CRL4 regulates the protein level of Dcx (A and B) Immunoblots of CRL4 and Dcx proteins from Cul4a- and Cul4b-immunoprecipitated DIV5 lysate from DIV5 (A) and DIV10 neurons (B). (C) Representative images of the PLA signals of Cul4a and Cul4b with Dcx and immunolabeled signal of Tau in DIV2 neurons (Scale bar, 30 μm). (D) Immunoblots and relative levels of Dcx protein normalized by the Actb level of DN-Cul4a and/or DN-Cul4b overexpressed PC12 cells (n = 4; ∗p < 0.05 to control group by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (E and F) Immunoblots of input (E) and immunoprecipitants by Dcx and Ubiquitin (Ub) (F) in control, Ddb1+Cul4a- Ddb1+Cul4b-expressing PC12 cells treated by MG132. Arrows and arrowheads indicate high-molecularly shifted and native Dcx, respectively. (G) Representative images of the PLA signals between Dcx and Crbn with phalloidin-labeled actin-filament in DIV4 neurons (Scale bar, 30 μm). (H) Immunoblots and relative levels of Dcx protein normalized by the Actb level in Cul4a- or Cul4b-overexpressing PC12 cells with or without Crbn (n = 3; ∗p < 0.05 to Mock group by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (I) Immunoblot and relative levels of Dcx protein normalized by the Actb level in Cul4a- or Cul4b-overexpressing PC12 cells with sgLacZ or sgCrbn (n = 3; ∗p < 0.05 to Mock group by two-tailed unpaired t-test; Data are represented as mean ± SEM.). (J and K) Immunoblots and relative levels of acetylated α-Tubulin (acetyl-Tub) normalized by the Actb level in Cul4a+Ddb1-or Cul4b+Ddb1-overexpressing PC12 cells with or without Crbn (J) (n = 3; ∗p < 0.05 to empty vector-transfected group by two-tailed unpaired t-test), and DN-Cul4a- or DN-Cul4b-expressing PC12 cells (K). (n = 4; ∗p < 0.05 to control group by two-tailed unpaired t-test). Data are represented as mean ± SEM. (L) Schematic summary of CRL4-mediated Dcx regulation.

Article Snippet: For genetic perturbation of CRL4, pcDNA3-HA2-CUL4A, pcDNA3-myc3-CUL4B, and pcDNA3-HA2-DDB1 were gifts from Yue Xiong (Addgene plasmid #19907, #19922, #19909).

Techniques: Western Blot, Immunoprecipitation, Immunolabeling, Control, Two Tailed Test, Ubiquitin Proteomics, Expressing, Labeling, Plasmid Preparation, Transfection

Journal: Molecular Cell

Article Title: The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma

doi: 10.1016/j.molcel.2020.12.046

Figure Lengend Snippet:

Article Snippet: pcDNA3-HA2 CaUL4A , Addgene , Cat.#: 19907 RRID:Addgene_19907 , .

Techniques: Generated, Control, Virus, Isolation, Recombinant, Transfection, Clone Assay, shRNA, Software