Journal: Nature Communications

Article Title: Molecular insights into degron recognition by CRL5 ASB7 ubiquitin ligase

doi: 10.1038/s41467-024-50556-x

Figure Lengend Snippet: a GST pull-down assays using GST-tagged GIGYF1, CCDC17, CEP152, CLIP2, and LZTS1 peptides to pull-down purified ASB7-Elongin B/C. Source data are provided as a Source Data file. Representative image, n = 3. b – f ITC titration and binding curves of purified ASB7-Elongin B/C with different degrons. Binding affinities ( K D ) are indicated. Source data are provided as Source Data file.

Article Snippet: The human ASB7 (residues 11–318) was amplified via PCR from a human cDNA library and seamlessly cloned into the pET28-MKH8SUMO vector (Addgene plasmid #79526).

Techniques: Purification, Titration, Binding Assay

Journal: Nature Communications

Article Title: Molecular insights into degron recognition by CRL5 ASB7 ubiquitin ligase

doi: 10.1038/s41467-024-50556-x

Figure Lengend Snippet: a Overall structure of ASB7-Elongin B/C bound to the LZTS1-degron peptide. ANK repeats, SOCS box, Elongin B and Elongin C are colored in pale green, lime green, light bule, and light pink, respectively. LZTS1-degron is shown in yellow. b Electrostatic potential surface of ASB7 bound to LZTS1-degron. Red, negative; Blue, positive. The helix-binding groove is ~30 Å long and ~9 Å wide. c Sequence alignment of ASB7 substrate degrons. The secondary structure and residues involved in site 1, site 2, and site 3 are indicated. d The α-helical LZTS1-degron is characterized three sites. Site 1, comprising five residues (yellow), is buried at the hydrophobic bottom of the binding groove. Site 2, comprising five residues (green), resides at the solvent-exposed surface on one side of the positively charged groove. Site 3 (cyan) docks on the other side of the negatively charged groove. e Cross-section view of the three different sites.

Article Snippet: The human ASB7 (residues 11–318) was amplified via PCR from a human cDNA library and seamlessly cloned into the pET28-MKH8SUMO vector (Addgene plasmid #79526).

Techniques: Binding Assay, Sequencing, Solvent

Journal: Nature Communications

Article Title: Molecular insights into degron recognition by CRL5 ASB7 ubiquitin ligase

doi: 10.1038/s41467-024-50556-x

Figure Lengend Snippet: a – d Close-up view of the interactions of E1, E8, L5, L12, and L16 in degron site 1 (yelloworange) with ASB7 (palegreen). The hydrogen bonds are shown as black dashed lines. e , f Close-up view of the interactions of L2, Q9, and E13 in site 2 with ASB7. g Interactions of K15 in site3 with ASB7. h Interactions of the extended Y19 of the LZTS1-degron with ASB7. i Schematic of the detailed interactions between ASB7 proteins and LZTS1-degron. Dashed lines and arrows indicate hydrogen bonds and hydrophobic interactions, respectively.

Article Snippet: The human ASB7 (residues 11–318) was amplified via PCR from a human cDNA library and seamlessly cloned into the pET28-MKH8SUMO vector (Addgene plasmid #79526).

Techniques:

Journal: Nature Communications

Article Title: Molecular insights into degron recognition by CRL5 ASB7 ubiquitin ligase

doi: 10.1038/s41467-024-50556-x

Figure Lengend Snippet: a ITC fitting curves of LZTS1 peptide titrated to wild-type ASB7 and site 1 mutants. NB, no apparent binding under our experimental conditions. b ITC fitting curves of LZTS1 peptide titrated to wild-type ASB7 and site 2/3 mutants. c ITC fitting curves of LZTS1 and substituted peptide titrated against wild-type ASB7. d ITC fitting curves of LZTS1 peptides of varying lengths against wild-type ASB7. The corresponding peptide lengths, sequences, and binding affinities are indicated. Source data are provided as Source Data file.

Article Snippet: The human ASB7 (residues 11–318) was amplified via PCR from a human cDNA library and seamlessly cloned into the pET28-MKH8SUMO vector (Addgene plasmid #79526).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Molecular insights into degron recognition by CRL5 ASB7 ubiquitin ligase

doi: 10.1038/s41467-024-50556-x

Figure Lengend Snippet: a – f Stability analysis of the GFP-fused LZTS1-degron was performed in ASB7 knockout HEK293T cells with overexpression of exogenous gASB7-resistant wild-type and mutant ASB7. The GFP/DsRed ratio was analyzed by flow cytometry. g Western blot analysis of Flag-tagged WT and mutant ASB7 expression in GPS reporter cell lines. Source data are provided as a Source Data file. Representative image, n = 3. h – m Stability analysis of GFP-fused CEP152-degron with overexpression of exogenous gASB7-resistant wild-type and mutant ASB7 in ASB7 knockout HEK293T cells. n Stability comparison of GFP-fused LZTS1-degron with substitutions in contact site 1, monitored by global protein stability assay. Source data are provided as Source Data file.

Article Snippet: The human ASB7 (residues 11–318) was amplified via PCR from a human cDNA library and seamlessly cloned into the pET28-MKH8SUMO vector (Addgene plasmid #79526).

Techniques: Knock-Out, Over Expression, Mutagenesis, Flow Cytometry, Western Blot, Expressing, Comparison, Stability Assay

Journal: Aging (Albany NY)

Article Title: MicroRNA 27b promotes cardiac fibrosis by targeting the FBW7/Snail pathway

doi: 10.18632/aging.102465

Figure Lengend Snippet: miR-27b modulated the expression of Snail by inhibiting its degradation ( A , B ) The mRNA level of Snail in CFs treated with AngII combined with miR-27i ( A ) or miR2-7b ( B ). ( C ) The effect of miR-27b on the expression of Snail in CFs treated with cycloheximide (CHX, 1 μg/ml) at indicated time points. ( D ) Roles of miR-27b in Snail ubiquitination. ( E ) Characterization of the messenger RNA (mRNA) of FBW7, which depicted miR-27b binding site (BS) in its 3′-untranslated region (UTR). ( F ) The interaction of FBW7 and Snail in CFs. ( G ) The effect of FBW7 overexpression on the expression of Snail in CFs treated with cycloheximide (CHX, 1 μg/ml) at indicated time points. ( H ) The effect of FBW7 overexpression on the ubiquitination of Snail. Arrow indicates the GFP-FBW7. Data were represented as mean ± SEM (n=4). N, p >0.05.

Article Snippet: The 3′UTR of the FBW7 luciferase reporter plasmid (pRL-TK 3′FBXW7 UTR) were obtained from Addgene (#26649).

Techniques: Expressing, Ubiquitin Proteomics, Binding Assay, Over Expression

Journal: Aging (Albany NY)

Article Title: MicroRNA 27b promotes cardiac fibrosis by targeting the FBW7/Snail pathway

doi: 10.18632/aging.102465

Figure Lengend Snippet: FBW7 was the miR-27b target in cardiac fibrosis ( A – C ) The mRNA levels of FBW7 in serum-treated CFs ( A ), AngII ( B ), or miR-27b ( C ). ( D ) Snail expression in CFs transfection with miR-27b and/or peGFP-FBW7 plasmids. ( E , F ) The proliferation of CFs treated in ( D ) were analyzed by MTT test ( E ), and BrdU assay ( F ). ( G ) CFs transfected with miR-27i and/or FBW7 siRNA were subjected to AngII treatment as indicated. Snail expression was analyzed using WB. ( H , I ) The proliferation of CFs treated in ( G ) was analyzed by MTT test ( H ), and BrdU assay ( I ). Data were represented as mean ± SEM (n=4). *, p <0.05; **, p <0.01.

Article Snippet: The 3′UTR of the FBW7 luciferase reporter plasmid (pRL-TK 3′FBXW7 UTR) were obtained from Addgene (#26649).

Techniques: Expressing, Transfection, BrdU Staining

Journal: Aging (Albany NY)

Article Title: MicroRNA 27b promotes cardiac fibrosis by targeting the FBW7/Snail pathway

doi: 10.18632/aging.102465

Figure Lengend Snippet: Antagomir-27b attenuated cardiac fibrosis in rat model of MI ( A ) Real-time PCR results of miR-27b levels in miR-27i or saline-treated specimens (3 weeks post-injection). ( B , C ) Analytical results of miR-27i-treated peri-infarct area of rat heart (3 weeks post-treatment). ( B ) Typical heart sections after treatments of Masson trichrome staining, laminin and collagen I immunostaining. Scale bar, 20 μm. ( C ) The percentage of tissue area represented the deposition of collagen I, where the automated image analyzer was used for its quantification. ( D ) Quantitative reverse transcription–PCR results of collagen I, collagen III, and MMP-9 mRNA levels. ( E ) FBW7 and Snail expression in rat heart. Data were represented as mean ± SEM (n=6). *, p <0.05; **, p <0.01.

Article Snippet: The 3′UTR of the FBW7 luciferase reporter plasmid (pRL-TK 3′FBXW7 UTR) were obtained from Addgene (#26649).

Techniques: Real-time Polymerase Chain Reaction, Saline, Injection, Staining, Immunostaining, Reverse Transcription, Expressing

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a Preassembled GST-IQGAP1-DBR and His 6 -DIAPH1-NT complexes were incubated with MBP-CLIP170-NT. In parallel preassembled GST-IQGAP1-DBR and MBP-CLIP170-NT complexes were incubated with His 6 -DIAPH1-NT. After reisolating GST-IQGAP1-DBR, the amount of co-purifying MBP-CLIP170-NT or His 6 -DIAPH1-NT was determined. NT = N-terminus, DBR = Diaphanous Binding Region. b Schematic representing inferred IQGAP1 interaction dynamics.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Incubation, Binding Assay

Journal: Nature Communications

Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

doi: 10.1038/s41467-021-22677-0

Figure Lengend Snippet: a Time-lapse images of GFP-CLIP170 during cell division. b Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 (top), IQGAP1 to CLIP170 (middle) and CLIP170 to DIAPH1 (bottom) during mitotic progression. c Quantitation of PLA foci in metaphase and anaphase * p < 0.0001 using a nonparametric two-tailed Mann-Whitney t -tests for three experimental repeats, n = 50 cells examined over three independent experiments. d Proximity ligation assays (PLA) mapping the proximity DIAPH1 to IQGAP1 and IQGAP1 to CLIP170 in Centrinone B-treated cells. e Quantitation of PLA foci in d . At cell poles lacking astral microtubules, DIAPH1 and IQGAP1 are in close proximity but IQGAP1 and CLIP170 are not. * p = 0.0006, ** p < 0.0001 using nonparametric two-tailed Mann-Whitney t -tests for three experimen t al repeats, n = 50 cells examined over three independent experiments. Scale bars are 10 μm.

Article Snippet: The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table .

Techniques: Ligation, Quantitation Assay, Two Tailed Test, MANN-WHITNEY