Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: (A) Domain organization of RNF168 indicating key functional domains and a degenerate PIP-like sequence residing in a disordered C-terminal region. The protein disorder profile was generated using the Protein Disorder Prediction (PrDOS) tool at http://prdos.hgc.jp/cgi-bin/top.cgi . (B) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and RNF168 WT or RNF168 ΔDPIP. 24 h post infection, some cultures were treated with HU (2mM, 2 h), camptothecin (CPT, 100 nM, 2 h), ATM inhibitor KU55933 100 nM, 2 h), or the WEE1 inhibitor MK1775 (10 μM, 2h). Chromatin extracts from the treated and untreated (control) cells were normalized for protein content and immunoprecipitated with anti-HA antibodies. Anti-HA immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (C) Replicate plates of RNF8 -/- U2OS cells were transiently infected with adenovirus vectors encoding FLAG-RNF168 WT, FLAG-RNF168 ΔDPIP, or FLAG-RNF168 ΔMIU2, or with a control ‘empty’ adenoviral vector. 48 h post-infection chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibodies. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (D) Isothermal titration calorimetry (left panel) was conducted by titrating synthetic peptide (p21, GRKRR QTSMTDFY HSKRRLIFS-amide where underlined residues denote PIP box; syringe, 150 - 300 μM) into a solution of PCNA (cell; 30 μM) in TBS. Control experiments using peptide injected into buffer alone showed minimal heats with no evidence of titration. Analysis of the isotherm yielded Kd = 76.3 nM and stoichiometry = 0.93 (Microcal Origin software). Replicate plates of H1299 cells were infected with adenovirus vectors encoding FLAG-RNF168 WT and HA-PCNA (or an ‘empty’ adenovirus vector for control). 36 h post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibody in the presence of different concentrations of peptides corresponding to the p21 or Polη PIP boxes. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies (right panel) . (E) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and FLAG-RNF168 WT or HA-PCNA and RNF168 ΔDPIP. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA (for control). 36 h post-infection, some plates were treated with 2 mM HU for 2 h. Chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody in the presence or absence of the p21 PIP box peptide (1 mM). Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (F) Replicate plates of H1299 cells were infected with adenovirus vectors encoding wild-type PCNA (HA-PCNA WT), ubiquitylation-resistant PCNA (HA-PCNA K164R), or a PCNA-ubiquitin fusion (HA-PCNA-Ub) in combination with FLAG-RNF168 WT adenovirus. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA viruses(for control). 36 h post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody. Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (G) H1299 cells were transiently co-transfected with expression constructs encoding HA-PCNA and FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. 48 h post-transfection cells were fixed, then stained with anti-HA and anti-FLAG antibodies prior to analysis by immunofluorescence confocal microscopy. The photographs are of representative cells co-expressing HA-PCNA and WT or ΔDPIP forms of FLAG-RNF168. The bar chart shows enumeration of cells containing HA-PCNA-co-localizing FLAG-RNF168 foci. Each data point represents the mean of results from 3 separate experiments and the error-bars represent the standard deviation. (H) RNF168 -/- U2OS cells were infected with adenoviral vectors encoding FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. 24 h post-infection cells were fixed and subject to proximity ligation assays to compare proximities of WT and ΔDPIP RNF168 with endogenous PCNA. The photographs are of representative DAPI-stained nuclei from each experimental condition. The bar chart showing enumeration of cells containing PCNA / FLAG-RNF168 PLA foci. Each data point represents the mean of results from 3 separate experiments and the error-bars represent the standard deviation. Ordinary one-way ANOVA demonstrated statistically significant difference between RNF168 WT and PIP (p = 0.0049).

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Functional Assay, Sequencing, Generated, Infection, Control, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, Isothermal Titration Calorimetry, Injection, Titration, Software, Ubiquitin Proteomics, Transfection, Expressing, Construct, Staining, Immunofluorescence, Confocal Microscopy, Standard Deviation, Ligation

Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: (A) Replicate plates of RNF8 +/+ and RNF8 -/- U2OS cells were infected with adenovirus vectors encoding RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157, or with an empty adenovirus vector as control. After 23 h, one of the empty vector control plates was irradiated with UVC (60 J/m 2 ). All plates of cells were collected 24 h post-infection and fractionated to give chromatin and soluble extracts. Cell extracts were normalized for protein content, resolved by SDS-PAGE and transferred to nitrocellulose membranes prior to immunoblotting with the indicated antibodies. (B) Replicate cultures of U2OS cells were infected with adenovirus vectors encoding RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157, or with an empty adenovirus vector as control. After 23 h cells were labelled with EdU, extracted with nonionic detergent to remove unbound MCM, fixed, and stained with anti-MCM2 (a marker for the MCM2-7 complex), PI (total DNA), and for EdU incorporation (active DNA synthesis). Cell cycle phases are defined by DNA content (PI-A) and DNA synthesis (Edu-A) in the upper plots. Nuclei containing loaded MCM2 in G1 and S phase are represented by blue and orange dots respectively. G1/G2/M phase cells negative for chromatin-loaded MCM2 are shown in grey. (C) Replicate plates of RAD18 +/+ and RAD18 -/- H1299 cells were infected with adenovirus vectors encoding different RNF168 variants (RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157) in combination with RAD18 adenovirus, or with an empty adenovirus vector as control. After 22 h, two cultures were irradiated with UVC (60 J/m 2 ). All plates of cells were collected 24 h post-infection and fractionated to give chromatin and soluble extracts. Cell extracts were normalized for protein content, resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with the indicated antibodies. (D) Replicate plates of RAD18 +/+ and RAD18 -/- H1299 cells were sequentially transfected with HLTF-directed siRNA or with non-targeting control siRNA (siCon), then with CMV-FLAG RNF168 WT (or with an empty vector for control). 48 h post-transfection, some cultures were conditionally irradiated with UVC (20 J/m 2 ). After 2 h chromatin fractions were prepared and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (E) Purified PCNA substrate was incubated in vitro with recombinant RNF168 and recombinant UBCH5 individually or in combination, with recombinant RAD18-RAD6 complex, or with a combination of RAD18-RAD6 complex and RNF168 in the presence of E1, ubiquitin and an ATP-regenerating system. Reactions were terminated after 15 min or 30 min and products were separated on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Infection, Plasmid Preparation, Control, Irradiation, SDS Page, Western Blot, Staining, Marker, DNA Synthesis, Transfection, Purification, Incubation, In Vitro, Recombinant, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: RNF168 and RAD18 both ubiquitinate histone H2A in the vicinity of DSB to promote 53BP1 signaling and NHEJ (left panel) and also ubiquitinate PCNA to promote TLS (right panel) . RAD18 additionally acts as a molecular chaperone for the RAD51D recombinase and promotes HR independently of its ubiquitin ligase activity (middle) . See ‘Discussion’ for details.

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Ubiquitin Proteomics, Activity Assay

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet:

Article Snippet: recombinant human 6His-Ube1 , RD System , Cat# E-305.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Luciferase, Mutagenesis, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software, Lysis

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 3 PAI-1 derived from endometrial stromal cells promotes senescence of ECs. A Analysis of interactions between various cells. B Analysis of interactions between PAI-1 + ESC and other various cells. C Immunofluorescence staining of PAI-1 (green) and vessels (red) in IUA patients (n = 5). scale bar = 100 μm. The supernatant from PAI-1+ ESCs was collected to stimulate HUVECs. D–J. Subsequently, the mRNA levels of P16 and P21 were analyzed by qPCR (n = 12) (D); the protein level of P21 was assessed by western blotting (n = 3) (E); the mRNA and protein levels of IL-6 were examined by qPCR (n = 6) and western blotting (n = 3), respectively (F); and the protein level of eNOS was determined by western blotting (n = 3) (G). SA-β-gal staining was observed (n = 3). scale bar = 100 μm (H), HUVECs migration was assayed (n = 3). scale bar = 100 μm (I), and tube formation was examined (n = 3) (J). scale bar = 100 μm.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Derivative Assay, Staining, Western Blot, Migration

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 4 PAI-1 promotes endothelial cell senescence through uPAR. A Immunofluorescence staining of uPAR (green) and CD31 (red) in endometria from IUA patients (n = 12) and controls (n = 12). Quantized co-expression area (%) was showed at right. scale bar = 100 μm. B, C The mRNA (n = 12) and protein levels (n = 3) of uPA in PAI-1+ESCs. D, E The mRNA (n = 11) and protein levels (n = 3) of uPAR in HUVECs treated with PAI-1+ESCs supernatant. F, G The mRNA and protein levels of P21 (n = 3) in HUVECs treated with Dox and transfected with si- PLAUR.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Staining, Expressing, Transfection

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 5 TGF-β upregulates PAI-1 in SMAD dependent manner. A The mRNA levels of PAI-1, ACTA2, and COL1A1 were examined by qPCR in ESCs after treatment with 10 ng/ml TGF-β for 3, 6, 12, 24 h respectively (n = 3). B The protein levels of PAI-1, ACTA2, and COL1A1 were tested by western blotting in ESCs after treatment with 10 ng/ml TGF-β for 6, 12, 24 h respectively (n = 3). C The mRNA level of PAI-1 were examined by qPCR in ESCs after treatment with 10 ng/ml TGF-β with or without SB-421542 (n = 3). D The protein levels of PAI-1, p-SMAD2, and SMAD2 were tested by western blotting in ESCs after treatment with 10 ng/ml TGF-β with or without SB-421542 (n = 3).

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Western Blot

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 6 PAI-1 inhibitors can inhibit endothelial senescence and endometrial fibrosis in mice. A Masson’s trichrome staining and immunohistochemical staining for COL1A1, α-SMA, PAI-1, uPA, and uPAR were performed on endometrial samples from normal controls, both with and without TPX, as well as in the IUA mouse model, both with and without TPX (n = 6). scale bar = 100 μm. B Immunofluorescence staining was conducted to detect P21 (green) and CD31 (red) in endometrial samples from normal controls, both with and without TPX, and in the IUA mouse model, both with and without TPX (n = 6). scale bar = 100 μm. The quantized results analyzed by ImageJ showed on right, each bar represents the mean ± SEM. ANOVA test, **p < 0.001, ***p < 0.0001.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Staining, Immunohistochemical staining

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 7 Schematic diagram illustrating how PAI-1 derived from ESCs promotes endothelial aging, leading to endometrial fibrosis. Trauma or infection elevates TGF-β in the endometrial microenvironment. TGF-β promotes the expression of PAI-1 in ESCs by activating SMAD2. PAI-1 synergizes with uPA to act on the uPAR receptor on endothelial cells, accelerating cellular senescence. Following endothelial cell senescence, the cells’ angiogenic capacity is inhibited, and they contribute to endometrial fibrosis by releasing factors such as SASP.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Derivative Assay, Infection, Expressing

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: The investigated serpins reduce SARS-CoV-2 infection by inhibition of TMPRSS2-mediated spike protein cleavage. (A) HEK-293T cells transfected with the indicated expression plasmids for 24 h were infected with SARS-CoV-2 (MOI = 0.1) for 6 h, and viral RNA was measured by qPCR. (B and C) Posttransfection (24 h) HEK293T cells were infected for 2 h (MOI = 1) and then trypsinized, washed with PBS, and lysed, and the RNA was extracted. Levels of viral RNA were quantified from cDNA synthesized with (B) random hexamers (C) or only the forward primer selectively quantifying the negative sense RNA. Data are cumulative of three independent experiments performed in triplicate; mean and SEM are shown, and statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Surface plasmon resonance analysis of TMPRSS2 binding to individual serpins. A 2-fold dilution series of TMPRSS2 ranging from 125 nM down to 7.8 nM over immobilized SERPINE1 with results shown as response units (RU). Binding kinetics for all serpins are summarized to the right, including the natural target for SERPINE1, tissue plasminogen activator (tPA), as a positive control. (E) TMPRSS2-mediated S-protein cleavage in the presence or absence of individual serpins and the known protease inhibitor nafamostat mesylate. Data from three independent experiments were quantified, and a representative blot is shown. (F) The intensity of bands in panel E corresponding to cleaved S-protein was quantified using ImageJ (Fuji) and normalized to S-protein and TMPRSS2 control. Mean values and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (G) HBEC ALI cultures were preincubated apically with recombinant SERPINE1, SERPINA1, or SERPINC1 and infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side was quantified by qPCR at the indicated time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (H) The concentrations of apically released SERPINA1 and SERPINE2 from HBEC ALI cultures from both group high and group low were determined by ELISA. The apical secretions were collected at three time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (***, P < 0.001).

Article Snippet: Recombinant SERPINE1 protein (PAI-1, 1786-PI), SERPINA1 protein (A1AT, 1268-PI), SERPINC1 protein (ATIII, 1267-PI), and nafamostat mesylate (3081) were purchased from R&D Systems.

Techniques: Infection, Inhibition, Transfection, Expressing, Synthesized, SPR Assay, Binding Assay, Positive Control, Protease Inhibitor, Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: Single-cell RNA sequencing shows distinct expression patterns of serpins in primary lung cells. (A) Schematic illustration of cell types annotated in the single-cell RNA analysis of HBEC ALI cultures. (B) UMAP plot (bidimensional), colored by annotated cell clusters. (C) Coarse-grained graph showing mean cell cluster group expression for SERPINA1, SERPINE1, and SERPINE2. (D) Infection levels analyzed by qPCR and shown as fold increase over 12-h input. (E) Heatmap displaying the mean expression of SERPINA1, SERPINE1, and SERPINE2 upon SARS-CoV-2 infection (±, MOI = 0.05) in HBEC ALI cultures from a group high (H) and a group low (L) donor.

Article Snippet: Recombinant SERPINE1 protein (PAI-1, 1786-PI), SERPINA1 protein (A1AT, 1268-PI), SERPINC1 protein (ATIII, 1267-PI), and nafamostat mesylate (3081) were purchased from R&D Systems.

Techniques: RNA Sequencing, Expressing, Infection

Journal: Open Biology

Article Title: Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

doi: 10.1098/rsob.130213

Figure Lengend Snippet: PINK1-dependent phosphorylation of Parkin Ser 65 leads to activation of Parkin E3 ligase activity and multi-monoubiquitylation of Miro1. Wild-type (WT) ( a ) but not kinase-inactive (KI) ( b ) PINK1 activates wild-type Parkin E3 ligase activity leading to Miro1 multi-monoubiquitylation, an effect that is blocked by mutant Parkin Ser65Ala (S65A) ( c ). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris–HCl (pH 7.5), 0.1 mM ethylene glycol tetra-acetic acid (EGTA), 10 mM MgCl 2 , 0.1% 2-mercaptoethanol and 0.1 mM ATP) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP) and 2 μg of His-Sumo-Miro1. Reactions were terminated after 60 min by addition of SDS–PAGE loading buffer and resolved by SDS–PAGE. Miro1, ubiquitin, Parkin and PINK1 were detected using anti-SUMO, anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Representative of three independent experiments.

Article Snippet: Kinase assays were incubated at 30°C for 60 min followed by addition of ubiquitylation assay components and Mastermix to a final volume of 50 μl (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 and 2 μg 6xHis-Sumo-Miro1 (wild-type or point mutants) both purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP).

Techniques: Activation Assay, Activity Assay, Mutagenesis, Incubation, Ubiquitin Assay, Recombinant, Purification, SDS Page

Journal: Developmental cell

Article Title: Ari-1 regulates myonuclear organization together with Parkin and is associated with aortic aneurysms

doi: 10.1016/j.devcel.2018.03.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A 50 μL reaction mixture for KoiU2 ubiquitination contained 1μM human E1 recombinant protein (Enzo), 5μM UbcH7 (Boston Biochem), 2μM GST-tagged E3 ubiquitin ligase, 5μM T7-KoiU2, 5mM Mg-ATP (Enzo) 2.5μM biotinylated ubiquitin (Enzo) and 1mM DTT in 1× ubiquitination buffer (Enzo).

Techniques: Recombinant, Plasmid Preparation, Protease Inhibitor, Lysis, Transfection, SYBR Green Assay, Mutagenesis, Modification, shRNA