Fig. 1.
Human DCNL proteins function in…
Fig. 1.
Human DCNL proteins function in a nonredundant manner for Cul neddylation in vivo.…
Fig. 1.Human DCNL proteins function in a nonredundant manner for Cul neddylation in vivo. (A) HeLa cells were transfected with control siRNA (scramble) or siRNA duplexes that specifically down-regulate hUbc12, DCNL1, or DCNL2. After 72 h, the neddylation state of Cul1 and Cul3 was analyzed by immunoblotting, and the ratio between neddylated and unneddylated Cul protein was quantified by LI-COR and normalized to scramble siRNA controls. The mean ratio from 4 independent experiments is indicated (see Fig. S3 C for statistics). DCNL1 and DCNL2 comigrate and were detected with an antibody that recognizes both proteins (see Fig. S4 C ); mRNA levels were quantified by quantitative RT-PCR ( Fig. S3 A ). (B) HeLa cells were transfected with scramble control siRNA or DCNL3 siRNA duplexes, and the neddylation state of Cul1–4 was analyzed after 72 h as described in A. The corresponding statistical analysis and mRNA quantifications are listed in Fig. S3 B and C . (C) HeLa cells transfected with scramble (Left) or DCNL3-specific siRNA duplexes (Right) were analyzed after 5 days by indirect immunofluorescence and DAPI staining. Multinucleated and butterfly nuclei were quantified from 3 independent experiments (bar graph). (Bars: 5 μm.) (D) Extracts from cells depleted for DCNL3 or treated with scramble RNAi controls (from B) were analyzed by immunoblotting for the Cul3 substrate Nrf2. (E) HA-tagged DCNL3 or the DCNL3 DAD-patch mutant were overexpressed in HeLa cells together with Flag-tagged Cul3. Total cell extracts were analyzed by immunoblotting with specific antibodies.
Fig. 2.
DCNL3 directly binds Cul3 and…
Fig. 2.
DCNL3 directly binds Cul3 and hUbc12. ( A ) HeLa cell extracts were…
Fig. 2.DCNL3 directly binds Cul3 and hUbc12. (A) HeLa cell extracts were immunoprecipitated (IP) with specific DCNL3 antibodies or nonspecific control IgG and examined for associated Culs. An aliquot of total extract (input) and supernatants after immunoprecipitation were analyzed (Left). (B) The indicated ratio of purified His-DCNL3, His-DCNL1, and control His-DCNL-DAD mutants was incubated in vitro with Sf9 cell-expressed GST-Cul3/Rbx1 complexes. Bound proteins were visualized by immunoblotting with anti-His antibodies. Control input levels of DCNL1, DCNL3, and Cul3 are shown (Left and Lower). (C) HA-tagged wild-type and the DAD-patch mutant (D241A, A265R, D271A) of DCNL3 were immunoprecipitated (IP HA) from extracts prepared from stable 293T-Rex cell lines (input), and associated Cul3 was visualized with specific antibodies. (D) In vitro binding between recombinant 6×His-DCNL3 and GST-tagged hUbc12 or the ubiquitin-specific enzymes GST-Cdc34 and GST-UbcH7. The interaction was analyzed by immunoblotting with anti-His antibodies, while the input levels of the GST-tagged proteins were controlled by Ponceau S staining.
Fig. 3.
Membrane localization of DCNL3 depends…
Fig. 3.
Membrane localization of DCNL3 depends on lipid modifications of its N-terminal domain. (…
Fig. 3.Membrane localization of DCNL3 depends on lipid modifications of its N-terminal domain. (A) Schematic representation of DCNL3 highlighting the N-terminal domain of DCNL3 with the conserved membrane-targeting motif. The mutated amino acids in DCNL3-G2A and DCNL3-C4A-C8A used in the experiments are indicated. (B) COS-7 cells expressing wild-type or the indicated DCNL3-HA mutants were incubated with azido-myristate or azido-palmitate followed by immunoprecipitation with HA antibodies (HA). Conjugation of the lipid analogues to phosphine-biotin was detected by immunoblotting with neutravidin-HRP (NA-HRP). The membranes were treated for control with 0.1M Tris·HCl or 0.1 M KOH to remove thioester-linked palmitate but not N-linked myristate. Note that myristoylation at G2 is a prerequisite for palmitoylation of the Cys residues C4 and C8. (C) HeLa cells transfected with an HA-control plasmid (HA), HA-tagged DCNL2, wild-type, or the indicated DCNL3 mutants were analyzed by indirect immunofluorescence with HA-antibodies. (Bars: 5 μm.) (D) Whole-cell extracts were separated into membrane and cytosol fractions prepared from HeLa cells harboring an HA-control plasmid (lanes 1) or expressing DCNL3-HA (lanes 2), DCNL3-G2A-HA (lanes 3), or DCNL3-C4A-C8A-HA (lanes 4). The proteins were detected by immunoblotting with HA-antibodies. Antibodies specific for the following markers control the different fractions: Na+/K+ ATPase (plasma membrane), α-tubulin (cytosol), and HNRPC (heterogeneous nuclear ribonucleoprotein C, nucleus). One-tenth of the whole-cell extract was loaded compared with the cytosol and membrane fractions. (E) Whole cell, membrane, and cytosol fractions were prepared from HeLa cells, and the partitioning of endogenous DCNL3, DCNL1/2, Cul1, Cul3, and CAND1 was followed by immunoblotting with specific antibodies. The markers are described in D.
Fig. 4.
DCNL3 recruits endogenous Cul3 to…
Fig. 4.
DCNL3 recruits endogenous Cul3 to the plasma membrane promoting Cul3 neddylation. ( A…
Fig. 4.DCNL3 recruits endogenous Cul3 to the plasma membrane promoting Cul3 neddylation. (A) (Top) HeLa cells harboring an HA-control plasmid (HA) or expressing HA-tagged DCNL3, DCNL3-DAD, or DCNL3-G2A were analyzed by indirect immunofluorescence with HA-antibodies. (Middle) The localization of endogenous Cul3 was visualized with specific antibodies. (Bottom) The overlay pictures show DNA (DAPI, blue), DCNL (HA, red), and Cul (green) stainings. Arrows indicate the accumulation of Cul3 at plasma membranes. (Bars: 5 μm.) (B) HeLa cells were transfected with an HA-control plasmid (HA) or plasmids expressing HA-tagged wild type or the indicated DCNL3 mutants together with Flag-tagged Cul3. The neddylation state of Cul3 and DCNL3-HA expression was analyzed by immunoblotting. (C) HeLa cells were transfected with DCNL3-HA in the presence of different myristoylation and palmitoylation inhibitors (HMA and 2BP, respectively) or DMSO as a solvent control. Membrane localization of DCNL3 was detected by indirect immunofluorescence using HA antibodies. (Bars: 5 μm.) (D) The neddylation state of endogenous Cul3 in the presence of the myristoylation (HMA) and palmitoylation (2BP) inhibitors was quantified by LI-COR from immunoblots of 3 independent experiments. The ratio of neddylated over unnedddylated Cul3 in HeLa cells treated with the inhibitors was normalized to DMSO controls and the mean ratio is indicated ( Fig. S6 C for statistics). Immunoblotting for α-tubulin controls for equal loading.
Fig. 5.
An intact DCNL-binding site is…
Fig. 5.
An intact DCNL-binding site is required for Cul3 neddylation and function in vivo.…
Fig. 5.An intact DCNL-binding site is required for Cul3 neddylation and function in vivo. (A) The neddylation state of HA-tagged Cul3 and the Cul3-R756A and Cul3-K712R mutants expressed in HeLa cells was tested by immunoblotting of total cell extracts with HA antibodies. (B) Flag-Cul3 or Flag-Cul3-R756A were ectopically expressed in HeLa cells either together with a HA-control vector (HA) or a plasmid harboring DCNL3-HA. Cell extracts (input) were immunoprecipitated with HA-antibodies (IP HA) and associated Flag-Cul3 was detected with anti-Flag antibodies. (C) HA-Cul3, HA-Cul3-R756A, or an empty HA-vector control were expressed in HeLa cells and immunoprecipitated with HA-antibodies (IP HA; Upper). The association of endogenous Rbx1 was detected by immunoblotting (IP HA; Lower). Immunoblotting of an aliquot of the extract before immunoprecipitation controls for the expression of HA-Cul3 and HA-Cul3-R756A, respectively (Left). (D) Flag-Cul3 or Flag-Cul3-K712R were expressed in HeLa cells together with either an HA-control or DCNL3-HA, and coimmunoprecipitation was analyzed as described in B. (E) Extracts from 293T-Rex cells stably expressing HA-Cul3, HA-Cul3-R756A, or HA-Cul3-K712R were analyzed with HA-antibodies after induction with doxycycline (+) for 24 h. (F) 293T-Rex cells stably expressing HA-tagged Cul3, Cul3-R756A, or Cul3-K712R were grown for 10 days in the presence (+Dox) or absence (−Dox) of doxycycline. Cells were harvested onto coverslips by cytospin and analyzed for Cul3-like phenotypes by α-tubulin and DAPI staining. Multinucleated and butterfly nuclei were quantified from 3 independent experiments. (Bars: 5 μm.)
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