plasmids for human prpf8, cep78, edd, and ddb1  (GenScript corporation)

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) The graph provides an overview of the splicing process. PRPF8 is a core component of the U5 snRNP and the spliceosome. B) Ubiquitination and degradation of CP110 regulates ciliogenesis. CP110 caps distal end of the mother centriole and thereby inhibits ciliogenesis. EDD-DYRK2-DDB1 VprBP complex is constitutively located at the (sub)distal end of the mother centriole. CEP350 recruits CEP78, and CEP78 activates VPRBP and the EDD-DYRK2-DDB1 VprBP complex. Phosphorylation of CP110 by DYRK2 enables recognition of CP110, which is brought close to EDD that transfers ubiquitin to CP110. PRPF8 functions as receptor for ubiquitin chains of CP110. Ubiquitination causes CP110-disassembly and removal from mother centriole, initiating ciliogenesis. C) Summary of renal and extra-renal manifestations in patients with PRPF8 variants. The patient with the de novo PRPF8 R1681W variant displayed the most severe phenotype with multiple malformations. None of the patients presented with RP symptoms. D) Examples of sequence conservation of PRPF8 amino acids mutated in CAKUT. E) Protein domain structure of human PRPF8 showing the position of de novo variants (red), heterozygous CAKUT variants (black) and RP mutations (magenta). Green arrow depicts a missense mutation ( Prpf8 +/N1531S ) in a mouse model exhibiting a ciliopathy phenotype. PRO8NT: PRP8 N-terminal domain or Bromodomain; PROCN PRO8 central domain; RT reverse transcriptase domain; RNaseH-like Ribonuclease H domain; Jab1/MPN Jun activation domain-binding protein 1/Mpr1, Pad1 N-terminal domain; RP Retinitis Pigmentosa.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Variant Assay, Sequencing, Mutagenesis, Reverse Transcription, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) Exon and domain structure of human DYRK2 . Arrowheads show positions of heterozygous CAKUT variants. B) Alphafold-predicted 3D structure of the DYRK2–CEP78 complex. The red square in the PAE plot suggests a potential direct interaction. Variants are scattered and do not cluster at the interface. C) DYRK2 kinase activity assessed via NDEL1 phosphorylation. Immunoblotting shows comparable phospho-NDEL1S336 levels for all CAKUT variants relative to WT, indicating preserved kinase function. K251R served as a kinase-dead control; empty vector as negative control. D) Co-IP of DYRK2 variants with EDD, DDB1, and VprBP. Two variants (p.Arg326Cys, p.Arg326His) showed reduced complex formation (n=3).

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Plasmid Preparation, Negative Control, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) The graph provides an overview of the splicing process. PRPF8 is a core component of the U5 snRNP and the spliceosome. B) Ubiquitination and degradation of CP110 regulates ciliogenesis. CP110 caps distal end of the mother centriole and thereby inhibits ciliogenesis. EDD-DYRK2-DDB1 VprBP complex is constitutively located at the (sub)distal end of the mother centriole. CEP350 recruits CEP78, and CEP78 activates VPRBP and the EDD-DYRK2-DDB1 VprBP complex. Phosphorylation of CP110 by DYRK2 enables recognition of CP110, which is brought close to EDD that transfers ubiquitin to CP110. PRPF8 functions as receptor for ubiquitin chains of CP110. Ubiquitination causes CP110-disassembly and removal from mother centriole, initiating ciliogenesis. C) Summary of renal and extra-renal manifestations in patients with PRPF8 variants. The patient with the de novo PRPF8 R1681W variant displayed the most severe phenotype with multiple malformations. None of the patients presented with RP symptoms. D) Examples of sequence conservation of PRPF8 amino acids mutated in CAKUT. E) Protein domain structure of human PRPF8 showing the position of de novo variants (red), heterozygous CAKUT variants (black) and RP mutations (magenta). Green arrow depicts a missense mutation ( Prpf8 +/N1531S ) in a mouse model exhibiting a ciliopathy phenotype. PRO8NT: PRP8 N-terminal domain or Bromodomain; PROCN PRO8 central domain; RT reverse transcriptase domain; RNaseH-like Ribonuclease H domain; Jab1/MPN Jun activation domain-binding protein 1/Mpr1, Pad1 N-terminal domain; RP Retinitis Pigmentosa.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Variant Assay, Sequencing, Mutagenesis, Reverse Transcription, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) Growth of yeast containing different Prp8 variants at various temperatures. A strain harboring the de novo variant PRPF8 R1681W ( Prp8 R1753W ) is inviable at 37 °C. B) CAKUT variants do not rescue the prp28-1 cold sensitive phenotype at 16 °C. C) Prp8 variants do not suppress the U4-cs1 phenotype at 16 °C, however PRPF8 R1414H (Prp8 R1468H ) shows a weak suppression of U4-cs1 at 23°C. D) Multiple Prp8 variants show genetic interactions with brr2-1 . E) Schematic overview of the ACT-1CUP1 assay, including the locations of non-consensus substitutions. F-H) ACT1-CUP1 assay results for the A3C (panel F), BS-C (panel G), BS-G (panel H), and UUG reporters (panel I).

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Variant Assay

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: A) No difference between ability to increase Gli1 and Ptch expression in Prpf8 +/N1531S mouse embryonic fibroblast treated with SAG. B) GLI1 and PTCH1 expression of RPE-1 cells transfected with plasmids endoding CAKUT variants ( red = de novo , green = Prpf8 N1531S , grey =CAKUT heterozygous), and RP variants (magenta) . Cells were stimulated with SAG and GLI1 and PTCH1 expression analyzed by RT-qPCR, using endogenous PRPF8 mRNA and 18S RNA levels for normalization. P13L and S1722G serve as negative controls.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: Expressing, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CAKUT variants in PRPF8, DYRK2 , and CEP78 : implications for splicing and ciliogenesis

doi: 10.1101/2025.07.16.665151

Figure Lengend Snippet: RNA in situ hybridization analysis of Prpf8 expression on sagittal sections of mouse kidney (first row), transverse sections of the ureter (second row) and sagittal sections of the bladder (third row) of wildtype embryos from E11.5 to E18.5. Note that all sections were developed for the same time, except the ones from the last column (*) for which color development was prolonged to detect weak expression domains. n =4 for each stage and tissue. Size bars represent 100 µm. ble, bladder epithelium; blm, bladder mesenchyme; cl, cloaca; k, kidney; u, ureter; ue, ureteric epithelium; um, ureteric mesenchyme; us, ureteric stalk, ut, ureteric tip.

Article Snippet: WT and mutant plasmids for human PRPF8, CEP78, EDD, and DDB1 were obtained from GenScript.

Techniques: RNA In Situ Hybridization, Expressing

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: DDB1 and CRBN enhance ClC-2 protein degradation. ( A , B ) Co-immunoprecipitation of Myc-ClC-2 and Flag-DDB1 ( A ), as well as of Flag-ClC-2 and HA-tagged CRBN (HA-CRBN) ( B ), in HEK293T cells. Flag-DDB1 and HA-CRBN were recognized with α-Flag and the anti-HA antibody (α-HA), respectively. ( C ) Interaction of endogenous CRBN with ClC-2 in the rat brain. Whole brain lysates were immunoprecipitated with the anti-ClC-2 antibody (α-ClC-2), followed by immunoblotting of the immunoprecipitates with α-ClC-2 or the anti-CRBN antibody (α-CRBN). Co-immunoprecipitation of CRBN was achieved by using α-ClC-2 but not by rabbit IgG (α-IgG). l.c.: IgG light chain. ( D – E ) (Upper panels ) Representative immunoblots showing the effect of Flag-DDB1 ( D ) or HA-CRBN ( E ) co-expression on the Myc-ClC-2 protein level. Co-expression with the Flag or the HA vector was used as the control experiment. ( Lower panels ) Quantification of the relative ClC-2 protein levels. Values from the DDB1 or the CRBN co-expression groups ( hatched bars ) were normalized to those for the corresponding vector control ( clear bars ) (*, t test: p < 0.05; n = 7–10). ( F ) ( Upper panel ) Representative immunoblots showing the lack of an effect of Flag-DDB2 co-expression on the Myc-ClC-2 protein level. Co-expression with the Flag vector was used as the control experiment. ( Lower panel ) Quantification of relative ClC-2 protein levels ( t test: p > 0.05; n = 3). ( G ) Representative immunoblots showing the effect of shRNA knockdown of endogenous DDB1 (shDDB1) or CRBN (shCRBN) in HEK293T cells on Myc-ClC-2. Infection with shLacZ was used as the control experiment. The numbers on the immunoblot denote the relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Control, shRNA, Knockdown, Infection

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: DDB1 and CRBN reduce ClC-2 protein stability. Representative immunoblots showing the effect of vector ( A ), DDB1 ( B ), or CRBN ( C ) co-expression on ClC-2 protein turnover kinetics in HEK293T cells. Transfected cells were subject to different treatment durations (0 to 8 h) of the protein synthesis inhibitor cycloheximide (CHX). Protein densities were normalized to those of corresponding no-treatment controls at 0 h. The numbers on the immunoblot denote relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Western Blot, Plasmid Preparation, Expressing, Transfection

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: Endogenous expression of CUL4 E3 ligase in testes and Leydig cells. Representative immunoblots showing the endogenous expression of ClC-2 ( A ), CUL4A ( B ), CUL4B ( C ), DDB1 ( D ), and CRBN ( E ) in lysates prepared from mouse testes or cultured mouse Leydig cells. Approximately 30 µg of protein was loaded into each lane.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Expressing, Western Blot, Cell Culture

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: CUL4 and DDB1 promote the degradation of endogenous ClC-2. Representative immunoblots showing the effect of shRNA knockdown of CUL4A (shCUL4A) ( A ), CUL4B (shCUL4B) ( B ), or DDB1 (shCDDB1) ( C ) on endogenous ClC-2 expression in MA-10 cells. Infection with shLacZ was used as the control experiment. The numbers on the immunoblot denote the relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Western Blot, shRNA, Knockdown, Expressing, Infection, Control

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: Aldosteronism-associated gain-of-function alteration in ClC-2 proteostasis. ( A ) ( Upper panel ) Representative immunoblot comparing the protein expression of Myc-ClC-2 WT, aldosteronism-related G32D mutant, and leukodystrophy-related G511R mutant overexpressed in HEK293T cells. ( Lower panel ) Quantification of relative ClC-2 protein levels. Protein density was standardized as the ratio of the ClC-2 signal to the cognate tubulin signal. Values from the mutant groups ( hatched bars ) were then normalized to those for the corresponding WT control ( clear bars ). Asterisks denote a significant difference from the WT (*, t test: p < 0.05; n = 7–9). ( B ) ( Upper panel ) Representative immunoblot showing the effect of the 24-h treatment of 10 μM lenalidomide on the Myc-ClC-2 G32D mutant. ( Lower panel ) Quantification of the relative ClC-2 G32D protein levels (*, t test: p < 0.05; n = 7). ( C ) ( Upper panel ) Representative immunoblot showing the effect of HA-CRBN or Flag-DDB1 co-expression on the Myc-ClC-2 G32D mutant. ( Lower panel ) Quantification of the relative ClC-2 G32D protein levels (*, t test: p < 0.05; n = 4–6). ( D ) Representative immunoblot showing the enhanced surface expression of the Myc-ClC-2 G32D mutant, as well as its reduction by CRBN co-expression. Cell lysates from surface-biotinylated intact HEK293T cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ). The numbers on the immunoblot denote the relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Western Blot, Expressing, Mutagenesis, Control

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: Schematic model of endoplasmic reticulum (ER)-associated degradation of ClC-2. In this schematic diagram of the regulation of ClC-2 proteostasis by ER quality control, the scaffold protein CUL4A/B forms a protein complex with the adaptor protein DDB1 and the substrate receptor protein CRBN. CUL4A/B also interacts with the RING-finger protein ROC, which in turn recruits the E2 ubiquitin conjugating enzyme (E2) that transfers ubiquitin (Ub) for covalent linkage to a substrate protein. We propose that, through the direct interaction between CRBN and ClC-2, the CUL4A/B-DDB1-CRBN E3 ubiquitin ligase complex catalyzes the ubiquitination of misfolded ClC-2 proteins. Ubiquitinated ClC-2 is subsequently targeted for proteasomal degradation. Loss-of-function, leukodystrophy-causing mutations may instigate substantial protein misfolding, leading to enhanced degradation of mutant ClC-2 proteins. In contrast, gain-of-function aldosteronism-causing mutations appear to facilitate protein stability, thereby reducing proteasomal degradation of mutant ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide effectively promotes, whereas the cullin E3 ligase inhibitor MLN4924 significantly attenuates, proteasomal degradation of both ClC-2 WT and disease-associated mutant proteins.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Control, Ubiquitin Proteomics, Mutagenesis

Journal: Scientific Reports

Article Title: The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

doi: 10.1038/srep10667

Figure Lengend Snippet: (A) Biochemical demonstration of CLC-1 polyubiquitination in HEK293T cells. (Left) Representative immunoblots showing the effect of HA-tagged lysine-less ubiquitin (HA-Ub-K0) co-expression on Myc-CLC-1. (Right) Quantification of relative CLC-1 protein expression level. Standardized protein densities of the Ub-K0 co-expression group ( hatched bars ) were normalized to those for the corresponding HA-vector control ( clear bars ). Asterisks denote significant difference from the control (*, t -test: p < 0.05; n = 5-6). (B) CLC-1 polyubiquitination [CLC-1-(Ub)n] by HA-Ub was reduced by DN-CUL4A, but not DN-CUL3. Co-expression with the Flag vector was used as the control experiment. Cell lysates were immunoprecipitated (IP) with the anti-Myc antibody, and protein ubiquitination was recognized by immunoblotting (IB) the immunoprecipitates with the anti-HA antibody. Corresponding expression levels of CLC-1 and actin in the lysates are shown in the Input lane. In all cases hereafter, input represents about 10% of the total protein used for immunoprecipitation. (C) CLC-1 polyubiquitination by endogenous ubiquitin was disrupted in the presence of DN-CUL4A/B. Co-expression with the Flag vector was used as the control experiment. Protein ubiquitination was identified by immunoblotting the immunoprecipitates with the anti-ubiquitin (Ub) antibody. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in .

Article Snippet: Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15818-15823), pcDNA3-Myc human cullin 3/4A/4B (Addgene 19893, 19951, 19922), pcDNA3-HA wild-type and lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19918), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Korea).

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Control, Immunoprecipitation

Journal: Scientific Reports

Article Title: The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

doi: 10.1038/srep10667

Figure Lengend Snippet: Biochemical demonstration of the regulation of CLC-1 by CRBN in HEK293T cells. (A) (Top) Representative immunoblots showing the effect of Flag-DDB1 or HA-CRBN over-expression on Myc-CLC-1 protein. Co-expression with the Flag/HA vector was used as the control experiment. (Bottom) Quantification of relative CLC-1 protein expression level. Values from the DDB1/CRBN co-expression group ( hatched bars ) were normalized to those for the corresponding vector control ( clear bars ). Asterisks denote significant difference from the control (*, t -test: p < 0.05; n =6-12). (B) CLC-1 polyubiquitination [CLC-1-(Ub)n] by endogenous ubiquitin was enhanced by CRBN over-expression. Cell lysates were immunoprecipitated (IP) with the anti-Myc antibody. Protein ubiquitination was identified by immunoblotting the immunoprecipitates with the anti-ubiquitin (Ub) antibody. (C) Representative immunoblots showing the effect of shRNA knock-down of endogenous DDB1 or CRBN. The numbers denote the relative CLC-1/DDB1/CRBN expression level with respect to the control shRNA for GFP. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in .

Article Snippet: Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15818-15823), pcDNA3-Myc human cullin 3/4A/4B (Addgene 19893, 19951, 19922), pcDNA3-HA wild-type and lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19918), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Korea).

Techniques: Western Blot, Over Expression, Expressing, Plasmid Preparation, Control, Ubiquitin Proteomics, Immunoprecipitation, shRNA, Knockdown