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: bioRxiv

Article Title: The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia

doi: 10.1101/2020.07.09.190074

Figure Lengend Snippet: Identification of three isoforms of human ACE2. A.) Colony PCR analysis of recombinant clones derived from 3’ primers, specific for variants 1, 2 and X3; X1 and; X2 revealed the presence of distinct ACE2 isoforms. Two clonal isolates of each variant were analyzed further. B.) Transfection into cells of the cloned isoforms and western blot analysis reveal that they all express protein of approximately 120 kDa. Antibodies generated to either the N-terminus (ProSci 3227) or C-terminus (Proteintech 66699) of ACE2 give essentially identical results when used in western blot analysis. C.) DNA sequence analysis reveals that the three variants differ at the C-terminus in both sequence length as well as amino acid sequence. Amino acid sequence shown represents residues 733 to 805 of the full length ACE2 (variant 1,2).

Article Snippet: For our analysis of ACE2 protein we continued to use two different antibodies, one to the N-terminus (ProSci 3227) and the other to the C-terminus region (ProteinTech 66699).

Techniques: Recombinant, Clone Assay, Derivative Assay, Variant Assay, Transfection, Western Blot, Generated, Sequencing

Journal: bioRxiv

Article Title: The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia

doi: 10.1101/2020.07.09.190074

Figure Lengend Snippet: Demonstration of glycosylated modification of the three isoforms of ACE2. Clonal isolates identified (two each) as the three isoforms were subjected to digestion with PNGaseF. As illustrated PNGaseF treatment results in a shift in molecular weight from about 120 kilodaltons to about 100 kilodaltons.

Article Snippet: For our analysis of ACE2 protein we continued to use two different antibodies, one to the N-terminus (ProSci 3227) and the other to the C-terminus region (ProteinTech 66699).

Techniques: Modification, Molecular Weight

Journal: bioRxiv

Article Title: The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia

doi: 10.1101/2020.07.09.190074

Figure Lengend Snippet: Immunofluorescence microscopy of individual isoforms of ACE2. HEK293 cells were transfected with each of the isoforms of ACE2 and then processed for immunofluorescent microscopy. As can be seen in the figure above, there are distinct localizations of the isoforms. Green; N-terminus antibodies (Proteintech 66699, 1:500 dilution). Red; C-terminus antibodies (ProSci 3227, 1:250 dilution).

Article Snippet: For our analysis of ACE2 protein we continued to use two different antibodies, one to the N-terminus (ProSci 3227) and the other to the C-terminus region (ProteinTech 66699).

Techniques: Immunofluorescence, Microscopy, Transfection

Journal: bioRxiv

Article Title: The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia

doi: 10.1101/2020.07.09.190074

Figure Lengend Snippet: A.) Human nasal epithelial cells were grown under submerged (progenitor maintenance conditions) cell growth or subjected to air-liquid interface growth conditions. Cells were harvested at the indicated times and analyzed by immunoprecipitation followed by western blot analysis. As can be seen progenitor cells do not appear to express ACE2 protein and that the process of differentiation induces expression. PNGaseF treatment reveals that ACE2 exists as a glycoprotein. B.) To determine when ACE2 protein expression occurs during the differentiation process, HNECs were allowed to develop under ALI conditions. Cells were harvested at the indicated times and analyzed by immunoprecipitation and western blotting for ACE2 expression. As seen in the figure, ACE2 protein is apparent at day 2 and levels rise through days 4 and 6.

Article Snippet: For our analysis of ACE2 protein we continued to use two different antibodies, one to the N-terminus (ProSci 3227) and the other to the C-terminus region (ProteinTech 66699).

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: bioRxiv

Article Title: The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia

doi: 10.1101/2020.07.09.190074

Figure Lengend Snippet: ACE2 protein localization was accomplished by fluorescent immunohistochemistry. We chose two different antibodies, one to the N-terminus and the other to the C-terminal half of the protein. Although both antibodies displayed the same signals when used in our western blot analysis they display distinct and overlapping signals via fluorescent immunohistochemistry. We observed ACE2 located both in the basal cell compartment and the ciliated cell compartment, but minimal signal in the central region of tissue. The apical cytosol of ciliated cells contained a mixture of individual signals and overlapping signals from both ACE2 antibodies. Only the ACE2 C-term antibody displayed plasma membrane signal on ciliated cells. When we compared ACE2 IHC patterns between ALI tissues derived from a 34-year old female to a 33-year old male we observed similar patterns but with varying intensity suggesting possible variation in the abundance of ACE2 protein in the airway between individuals. (A) ACE2 immunohistochemistry. (B) Rabbit and Mouse isotype controls (C) Immunohistochemical stain identifying progenitor cells (basal cells) and the differentiated cell types.

Article Snippet: For our analysis of ACE2 protein we continued to use two different antibodies, one to the N-terminus (ProSci 3227) and the other to the C-terminus region (ProteinTech 66699).

Techniques: Immunohistochemistry, Western Blot, Clinical Proteomics, Membrane, Derivative Assay, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Design of quinoline SARS-CoV-2 papain-like protease inhibitors as oral antiviral drug candidates

doi: 10.1038/s41467-025-56902-x

Figure Lengend Snippet: a Differential scanning fluorimetry assay of Jun13296 in stabilizing SARS-CoV-2 PL pro . Jun12682 was included as a positive control for comparison. Data from Jun12682 is the mean of two repeats, and data from Jun13296 is the mean ± standard deviation of three technical repeats. b K i plot of Jun13296 in inhibiting SARS-CoV-2 PL pro hydrolysis of ISG15-AMC. c K i plot of Jun13296 in inhibiting SARS-CoV-2 PL pro hydrolysis of Ub-AMC. d Counter screening of Jun 13296 against host proteases USP2, USP7, USP8, USP14, USP15, USP30, UCH-L1, cathepsin B, cathepsin K, calpain-1, trypsin, and caspase 3. Data in ( d ) are presented as mean ± standard deviation of two technical repeats. Source data are provided as a file.

Article Snippet: E-520-025), 500 nM USP14 (ProSci, 91-171), 1 nM USP15 (R&D systems, E594), 20 nM USP30 (R&D systems, E582), and 1 nM UCH-L1 (R&D systems, 6007-CY).

Techniques: Fluorimetry Assay, Positive Control, Comparison, Standard Deviation