Journal: Cell Death & Disease

Article Title: Cellular senescence promotes macrophage-to-myofibroblast transition in chronic ischemic renal disease

doi: 10.1038/s41419-025-07666-1

Figure Lengend Snippet: SA-β-gal staining ( A ) and the mRNA expression of senescence markers (PAI-1, p16 INK-4a , p21 Cip1/Waf1 , p53, IL-6, MCP-1, TNF-α) ( B – H ) were significantly increased in human renal proximal tubular epithelial cells (HRPTEpiC) following TNF-α and TGF-β treatment. Similarly, the protein levels of IFITM3 were elevated under the same conditions ( I , J ). Data are mean ± SD ( n = 3/group). * P < 0.05 vs. Normal. PAI-1: plasminogen activator inhibitor-1. Scale bar: 200 µm ( A ).

Article Snippet: Firstly, human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA) [ , ] were grown in an epithelial cell medium (ScienCell) containing epithelial cell growth supplement (ScienCell), in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

Techniques: Staining, Expressing

Journal: Cell Death & Disease

Article Title: Cellular senescence promotes macrophage-to-myofibroblast transition in chronic ischemic renal disease

doi: 10.1038/s41419-025-07666-1

Figure Lengend Snippet: A Schematic of the in vitro experimental protocol. B , C : Immunofluorescence staining of Ki67 and quantitative analysis of Ki67 + cells. The number of Ki67 + cells decreased after co-culture with senescent human renal proximal tubular epithelial cells (HRPTEpiC) (senescent cells, SC). Scale bar: 100 µm ( B ). The data are mean ± SD (n = 3/group). D – H : The effects of IFITM-3 and ITGB-3 on macrophage senescence. IFITM3 and ITGB3 were manipulated in SC HRPTEpiC and macrophages, respectively. Macrophages were subsequently collected for Western blot analysis of senescence markers p21 Cip1/Waf1 , p53, p-p53.S15, and γ-H2AX. Silencing ITGB3 or IFITM3 individually using siRNA reduced the expression of these senescence markers. However, this blunting effect was mitigated when IFITM3 was overexpressed in HRPTEpiC, indicating that IFITM3 plays a critical role in maintaining senescence signaling despite ITGB3 knockdown. Data are mean ± SD ( n = 3/group). * P < 0.05 vs. Normal control (NC) and Non-SC groups; # P < 0.05 vs. SC group; and P < 0.05 vs. SC + IFITM3 over-expressing groups. I – K : Relative mRNA expression of the senescence markers p16 INK-4a , p21 Cip1/Waf1 , and p53 increased in macrophages co-incubated with senescent cells. β-actin was used as loading control. Data are mean ± SD ( n = 3/group). * P < 0.05 vs. Normal.

Article Snippet: Firstly, human renal proximal tubular epithelial cells (HRPTEpiC) (ScienCell, Carlsbad, CA) [ , ] were grown in an epithelial cell medium (ScienCell) containing epithelial cell growth supplement (ScienCell), in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C.

Techniques: In Vitro, Immunofluorescence, Staining, Co-Culture Assay, Western Blot, Expressing, Knockdown, Control, Incubation

Journal: Frontiers in microbiology

Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.

doi: 10.3389/fmicb.2021.713713

Figure Lengend Snippet: FIGURE 9 | Schematic representation of SARS-CoV-2 genome indicating the amplicons for the COVID-19 molecular diagnostics by RT-LAMP. Structural representation of SARS-CoV-2 virion shows the main particle parts. LAMP primer regions are indicated as previously reported (Baek et al., 2020; Ben-Assa et al., 2020; Butler et al., 2020; Chow et al., 2020; Dudley et al., 2020; Song et al., 2021; Ganguli et al., 2020; Huang et al., 2020; Lamb et al., 2020; Lu et al., 2020; Mohon et al., 2020; Park et al., 2020; Rabe and Cepko, 2020; Thi et al., 2020; Yan et al., 2020; Yu et al., 2020; Zhang et al., 2020a,b; Anahtar et al., 2021; Bhadra et al., 2021; Bokelmann et al., 2021; González-González et al., 2021). ORF, open reading frame; RdRp, RNA-dependent RNA polymerase; NSP, nonstructural protein. Schematic representation created using Snap Gene Viewer software version 5.0.7; N1, N2, and N3_CDC correspond to the amplicons for SARS-CoV-2 detection by RT-PCR. Created with biorender.com.

Article Snippet: 2019-SARS-CoV-2 Detection Reagent Kit (Eiken Chemical, Tokyo, Japan) Turbidity or fluorescence (+ calcein) 63◦C/18–60 min 100%/100% 20–110 copies/Rx 130 PLA General Hospital, Beijing, China Yan et al., 2020 iLACO Respiratory (not detailed) NI ORF1ab NI Yes NEB #M1800 Color 65◦C/ ≥20 min 89.9%/NI 10 copies/μL (detection threshold of 60 copies/μL); equivalent to 35–37 Ct in RT-PCR 248 Shenyang province, China Yu et al., 2020 Respiratory swabs (not detailed) VTM N gene and ORF1a NI Yes NEB #M1800 + SYTO-9 Color and fluorescence 65◦C/30 min NI 4.8 copies/μL 6 Wuhan Institute of Virology, China Zhang et al., 2020a Synthetic SARS-CoV-2 RNA NA N gene; E gene and As1e gene (ORF1a) Human actin B gene Yes NEB #M1800 + SYTO-9 Color and fluorescence 65◦C/20 min 87.5% 2 copies/μL NA NA Zhang et al., 2020b NEB #M1800, WarmStart Colorimetric RT-LAMP 2 × Master Mix; NEB #E1700, WarmStart LAMP kit (DNA and RNA); RdRp, RNA-dependent RNA polymerase (harbored by ORF1ab SARS-COV-2 genome region); NI, noninformed; NA, not applied; UTM, Universal Transport medium; VTM, viral transport medium, commonly containing, Hank’s balanced salt solution at pH 7.4 containing BSA (1%), amphotericin (15 μg/mL), penicillin G (100 units/mL), and streptomycin (50 μg/mL); Prot K, proteinase K; BD UVTM, Becton–Dickinson Universal Viral Transport Media system; SPS, sample preservation solution; QuantiFluor, Promega system for dsDNA quantification using a DNA intercalating dye; Prot.

Techniques: Software, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Reduced MDM2 protein in T47D cells causes reduced chromatin phosphoproteins 53BP1 and MDC1. ( A ) Experimental design workflow of the SILAC analysis (created in BioRender. Harmon, K. (2025) https://BioRender.com/oyd7ala ). Chromatin isolated from a mixture of T47D vector control cells (MDM2-competent) cultured in natural amino acid medium, and T47Dshmdm2 cells (MDM2-depleted) cultured in heavy isotope amino acid medium, was subjected to proteolysis followed by phospho-peptide purification and enrichment. Scatter plot represents the H/L ratio versus abundance of peptides identified by mass spectrometry, with those corresponding to TP53BP1 (magenta), TP53 (blue), MCM2 (green), and MDC1(brown) highlighted. ( B ) Chromatin (5 μg) isolated from T47D vector control, T47Dshmdm2, and T47Dshmdmx cells was subjected to SDS–PAGE/western blot analysis for 53BP1, MDC1, MCM4, lamin A/C, and mtp53. ( C and D ) IF of total 53BP1 (i), phospho-53BP1 ser25 (ii), or phospho- 53BP1 ser1778 (iii) within T47D vector control nuclei [(i) 528, (ii) 549, and (iii) 501], T47Dshmdm2 nuclei [(i) 549,(ii) 520, and (iii) 501], and T47Dshmdmx nuclei [(i) 623, (ii) 376, and (iii) 501]. Confocal images for six fields for each were acquired and the number of 53BP1 foci per nucleus from each cell population indicated above was determined. Representative data ( n = 3) with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Multiplex sample analysis, Isolation, Plasmid Preparation, Control, Cell Culture, Purification, Mass Spectrometry, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: 53BP1 in breast cancer cells interacts with both mtp53 and MDM2. ( A ) Relative abundance of mtp53, MDM2, MDMX, 53BP1, and MDC1 within whole cell extracts (20 μg) prepared from T47D (L194F) and MDA-MB-231 (R280K) cell lines determined by SDS–PAGE/western blot analysis. ( B–D ) Association of mtp53, MDM2, and 53BP1 in vivo measured using the PLA. PLA analyses of mtp53-53BP1 (panel B), MDM2-mtp53 (panel C), and MDM2-53BP1 (panel D) within T47D and MDA-MB-231 cells were performed as described in the “Materials and methods” section; primary antibodies are PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 3 for T47D, n = 2 for MDA-MB-231). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: Panel B vector (T47D = 276; MDA-MB-231 = 108), shmdmx (T47D = 239; MDA-MB-231 = 128), and shmdm2 (T47D = 310; MDA-MB-231 = 106); Panel C vector (T47D = 92; MDA-MB-231 = 104), shmdmx (T47D = 108; MDA-MB-231 = 131), and shmdm2 (T47D = 101; MDA-MB-231 = 136); Panel D vector (T47D = 761; MDA-MB-231 = 129), shmdmx (T47D = 529; MDA-MB-231 = 142), and shmdm2 (T47D = 964; MDA-MB-231 = 163). **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: SDS Page, Western Blot, In Vivo, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2–53BP1 interaction is promoted by the mtp53 C-terminus. ( A ) The C-terminus of mtp53 R273H within MDA-MB-468 was modified using CRISPR–Cas9 to create the cell line MDA-MB-468 R273Hfs347Δ360-393 (termed G6; mtp53 derivative R273HΔC). ( B ) Relative protein levels of 53BP1, MDM2, and mtp53 within MDA-MB-468 (25, 12.5, 6.25, and 3.125 μg) and G6 (25 μg) cell lines was examined by SDS–PAGE/western blot analysis. ( C ) Loss of mtp53 C-terminus disrupts mtp53 co-IP with MDM2. Extracts from MDA-MB-468 and G6 cell lines were incubated with either normal mouse IgG (negative control) or anti-MDM2 antibodies 4B2 (lanes 1–7) or SMP14 (lanes 8–14), and IP reactions were examined for MDM2 and mtp53 by western blot analysis. Lanes 1–7: 4B2 IP reactions from 800 μg of extract (input); lanes contain 10% of total IP and 2% of input. Lanes 8–14: SMP14 IP reactions from 1600 μg of extract; lanes contain 12.5% each IP and 0.5% of input. Lanes labeled 2× (lane 7 for the 4B2 IPs and lane 14 for the SMP14 IPs) contain twice the amount of the G6 extract MDM2 IP; a lighter exposure of mtp53 input is presented due to the vast excess of mtp53 compared to MDM2 within the MDA-MB-468 cell lines. ( D–F ) PLA analysis of mtp53–53BP1 (panel D), MDM2–mtp53 (panel E), and MDM2–53BP1 (panel F) in MDA-MB-468 and G6. PLA analysis of the indicated proteins was measured using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 2A9 antibodies. Confocal images for 3–5 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 2). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (D) R273H = 120; R273HΔC = 144; (E) R273H = 131; R273HΔC = 159; (F) R273H = 102; R273HΔC = 138. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, CRISPR, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Incubation, Negative Control, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2-mtp53 and MDM2–53BP1 interactions are Nutlin 3a sensitive. ( A ) Western blot analysis of whole cell extracts (10 μg) from MCF7 and T47D cell line populations treated for the indicated time with either vehicle or 10 μM Nutlin 3a (labeled N3a or “+” in graphs) for the indicated proteins. ( B ) Nutlin 3a does not inhibit T47D cell cycle progression. Twenty-four hour post-treatment with either vehicle or 10 μM Nutlin 3a, cells were labeled with EdU for 20 min and assayed for Cyclin A2 by immunofluorescence. Confocal images from at least three fields were acquired and the number of EdU + and Cyclin A + nuclei were quantified (tabulated in S4, panel C) in each population of vehicle-treated [vector = 289; shmdmx = 278; shmdm2 = 249] and Nutlin 3a-treated [vector = 227; shmdmx = 283; shmdm2 = 245] cells. The S/G2 fraction (total Cyclin A + nuclei) for vehicle-treated: vector = 33.6%, shmdmx = 37.8%, shmdm2 = 36.8%; Nutlin 3a-treated: vector = 38.3%, shmdmx = 27.0%, shmdm2 = 29.4%. ( C and D ) Nutlin 3a disrupts mtp53–MDM2 and 53BP1–MDM2 PLA foci in T47D. Twenty-four hour post-treatment with vehicle or 10 μM Nutlin 3a cell populations PLA interactions were measured between MDM2–mtp53 (panel C) and MDM2–53BP1 (panel D) using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2 primary antibodies. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus from each cell population was determined ( n = 2 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the materials and methods from the indicated number of cells in panel: (C) vehicle-treated [vector = 328; shmdmx = 298; shmdm2 = 249], Nutlin 3a-treated [vector = 323; shmdmx = 364; shmdm2 = 285] and (D) vehicle-treated [vector = 543; shmdmx = 548; shmdm2 = 455], Nutlin 3a-treated [vector = 610; shmdmx = 545; shmdm2 = 618]. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Western Blot, Labeling, Immunofluorescence, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: MDM2 promotes 53BP1–MDC1 complex formation. ( A ) Reduced 53BP1–MDC1 PLA foci in T47D lacking MDM2. EdU-labeled T47D vector and shmdm2 cells were assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Shown under representative images ( n = 3) is the number of EdU + nuclei identified within each cell population and graphed is the number of PLA foci per nucleus. For the PLA analysis representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: vector (total = 329, S-phase = 100, G1-G2 = 229); shmdm2 (total = 251, S-phase = 79, G1-G2 = 172). ( B ) Activation of the DDR in T47D cell lines by Etoposide but not Nutlin-3a. Whole cell extracts (20 μg) from T47D cell lines treated with either 50 μM Etoposide for 5 h (Etop), or 10 μM Nutlin 3a for 24 h (N3a) were analyzed for the indicated proteins by WB. ( C and D ) MDC1–53BP1 PLA foci are disrupted by both DDR activation and Nutlin 3a. MDC1–53BP1 PLA analyses were performed within each T47D cell line at the indicated time points post Etoposide treatment (panel C) or 24 h-post Nutlin 3a treatment (panel D) using PLA goat anti-53BP1 and PLA rabbit anti-MDC1. Confocal images for 3–6 fields for each were acquired and the number of PLA per nucleus from each cell population was determined ( n = 3 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (C) vehicle-treated (0 h Etop) [vector = 208; shmdmx = 198; shmdm2 = 247], 2 h Etoposide [vector = 205; shmdmx = 184; shmdm2 = 231], 5 h Etoposide [vector = 189; shmdmx = 224; shmdm2 = 176]; (D) vehicle-treated [vector = 198; shmdmx = 270; shmdm2 = 336] and Nutlin 3a-treated [vector = 380; shmdmx = 339; shmdm2 = 252]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Labeling, Plasmid Preparation, Activation Assay

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. ( A ) Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. 53BP1 was immunoprecipitated from T47D CES as described in the “Materials and methods” section subjected to western blot analysis for 53BP1, MDC1, MDM2, and p53. Lanes are as follows: (1) cytoplasmic extract, (2) CES IP input, (3) IP with IgG, and (4) IP for 53BP1. ( B ) Inhibition of ATM promotes MDM2 activity. Western blot analysis for the indicated proteins within extracts (10 μg) from T47D populations treated for 24 h with either vehicle,10 μM ALRN-6924 (MDM2/X dual inhibitor), 10 μM KU-55933 (ATMi; ATM inhibitor) or both. ( C ) The MDM2 inhibitor ALRN-6924 reduces whereas the ATM inhibitor increases MDC1–53BP1 PLA foci in T47D. Twenty-four hour post treatment with either vehicle,10 μM ALRN-6924, 10 μM KU-55933 (ATMi) or both T47D vector and shmdm2 cells were labeled with EdU for 20 min and then assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Confocal images for several fields were acquired and the number of PLA foci/nucleus from each cell population was determined. Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in: vehicle-treated [vector = 186; shmdm2 = 87], ATMi-treated [vector = 163; shmdm2 = 188], ALRN-6924-treated [vector = 196; shmdm2 = 176], ALRN-6924 + ATMi-treated [vector = 154; shmdm2 = 159]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Inhibition, Activity Assay, Plasmid Preparation, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Depletion of MDM2 increases poly(ADP-ribose) modification (PARylation) levels of chromatin bound proteins. Cytosolic ( A ) and chromatin ( B ) fractions were prepared from T47D cells with constitutive shmdm2, shmdmx, or mir30 shRNA-expressing vector cells treated with either vehicle (DMSO), or a combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h, or combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h and then replaced with fresh media for an additional 24 h. Ten micrograms of cytosolic or chromatin protein was loaded on a SDS–PAGE and protein levels were determined by western blot analysis using the indicated antibodies.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, shRNA, Expressing, Plasmid Preparation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: DISC1 interacts with mitochondrial trafficking complex proteins to regulate transport in dendrites in addition to axons. A , GFP trap co-immunoprecipitation experiments from COS7 cells show robust interaction of DISC1 with GFP Miro1, GFP Miro2, GFP TRAK1, and GFP TRAK2. B , proximity ligation assay in SH-SY5Y cells with DISC1 antibody or a DISC1 and Miro1 antibody shows significantly increased signal in the dual antibody condition over background, indicating that DISC1 and Miro1 interact within the cell ( n = 3 individual preparations). Scale bar = 20 μm. IB , immunoblot; IN , input. C and D , co-immunoprecipitation experiments with rat brain homogenate showing DISC1 to be part of a native complex with Miro ( C ) and TRAK1 ( n = 3) or TRAK2 ( n = 3) ( D ). E , example of live labeling of the axon initial segment ( AIS ) to distinguish axons and dendrites within the same neuron. F , kymographs showing movement of mitochondria through the axons and dendrites over time. Moving mitochondria are indicated by diagonal lines and stationary mitochondria by straight lines. G , dendritic compartments ( gray bars ) and axonal compartments ( black bars ) were assayed for mitochondrial movement, and the percentage of moving mitochondria was quantified with and without expression of DISC1 (dendrites: n = 15 control neurons and n = 16 DISC1 neurons, *, p = 0.02; axons: n = 16 control neurons and n = 13 DISC1 neurons, *, p = 0.02). Scale bar = 5 μm. NS , not significant.

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Immunoprecipitation, Proximity Ligation Assay, Western Blot, Labeling, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: DISC1 is recruited to mitochondria by components of the mitochondrial trafficking complex. A , immunocytochemistry in COS7 cells showing localization of exogenous DISC1 with and without myc Miro1 overexpression. Mitochondria are labeled with MtDsRed2. Scale bar = 20 μm. B , percentage of DISC1 on mitochondria shown in A ( n = 13–15 cells from three individual experiments, ***, p = 2.29 × 10 −5 ). Overexpression of myc Miro1 recruits DISC1 to mitochondria. C , mitochondrial fractionation from COS7 cells shows an increase in DISC1 in this compartment with GFP TRAK1 and myc Miro overexpression ( n = 3). IN , input.

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Immunocytochemistry, Over Expression, Labeling, Fractionation

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: The DISC1 N terminus mediates the interaction with Miro and TRAKs. A , schematic of the DISC1 protein showing domains present in deletion constructs used. Coiled-coil domains are dark blue , and nuclear import or export signals are dark red. B , schematic of TRAK2 showing coiled-coil domains ( dark blue ). C and D , mapping the region of DISC1 that interacts with myc Miro1 ( C ) and GFP TRAK2 ( D ). Co-IP experiments from COS7 cells show that the N-terminal 301 amino acids interact with Miro and TRAK2, whereas amino acids 313–854 are not pulled down. Arrowhead , highlights full-length ( FL ) HA DISC1 band; *, indicates nonspecific band. E and F , mapping the region of TRAK2 which interacts with DISC1. DISC1 interacts with full-length TRAK2 and TRAK2-(1–700) ( E ) but does not interact with the Miro binding domain ( F ). G , kymographs showing effect of overexpressing the DISC1-Miro binding domain on mitochondrial transport. Scale bar = 10 μm. H , percentage of moving mitochondria quantified in axons and dendrites. Overexpression of the DISC1-Miro binding domain prevents mitochondrial transport ( n = 23 ctrl and n = 21 DISC1-(1–301)-expressing neurons from three preparations, **, p = 0.002). I , quantification of percentage of moving mitochondria in axons and dendrites expressing MtDsRed2 (ctrl) or DISC1-(313–854), the region that does not interact with Miro1 ( n = 12 ctrl and 11 DISC1-(313–854)-expressing neurons from three preparations, p = 0.6). IB , immunoblot; IN , input; NS , not significant.

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Construct, Co-Immunoprecipitation Assay, Binding Assay, Over Expression, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: The DISC1-Boymaw fusion protein inhibits mitochondrial trafficking. A , immunocytochemistry in hippocampal neurons showing localization of the Boymaw protein. Scale bar = 20 μm, 5 μm on zoomed image ( right ). B , line scan of zoomed process showing that the DISC1-Boymaw fusion protein localizes to mitochondria in axons and dendrites. ( A.U. = arbitrary units.) C , kymographs showing mitochondrial transport in axons expressing MtDsRed2 and co-expressing HA Boymaw. Scale bar = 10 μm. D , percentage of moving mitochondria in neurons was quantified with and without expression of HA Boymaw. The presence of the HA Boymaw fusion protein inhibits mitochondrial trafficking ( n = 32 ctrl neurons and 26 HA Boymaw-expressing neurons, ***, p = 0.001). E and F , Boymaw expression has no impact on synaptophysin trafficking ( E ), as quantified in F ( n = 17 control and n = 19 HA Boymaw-expressing neurons from three preparations, p = 0.9). NS , not significant. Scale bar = 10 μm.

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Immunocytochemistry, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: DISC1-mediated mitochondrial trafficking is necessary for normal dendritic development. A , representative images showing control 10 DIV neurons and those expressing the DISC1-Miro binding domain (residues 1–301). GFP was used to visualize neuronal morphology. Scale bar = 10 μm. B , total dendritic length/cell (***, p = 0.001). C , Sholl analysis reveals a significant decrease in intersections at 80 and 100 μm from the soma (*, p < 0.05). D , average number of branch points/cell is decreased with DISC1-Miro binding domain expression (*, p = 0.04). E , Sholl analysis of branch points (*, p < 0.05 at 90 μm from the soma) ( n = 16 neurons from four preparations). F , representative images showing control 10 DIV neurons expressing GFP in the presence or absence of HA Boymaw. Scale bar = 10 μm. G , total dendritic length is decreased when the DISC1-Boymaw fusion protein is expressed (**, p = 0.004). H , Sholl analysis showing the number of intersections at 10-μm intervals. A significant decrease in intersections in Boymaw-expressing neurons is seen proximal to the soma at a distance of 50–80 μm away from the soma (*, p < 0.05 in each case). I , analysis of branch points reveals expression of the DISC1-Boymaw fusion protein to decrease in the number of branch points/cell (*, p = 0.03). J , Sholl analysis reveals a significant decrease 50 μm from the soma (*, p < 0.05, n = 15–16 neurons from four preparations).

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Control, Expressing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: DISC1 interacts with mitofusins. A and B , DISC1 1–301 decreases the length of mitochondria ( A ) as quantified in B (ctrl = 2. 1 μm ± 0.065, DISC1-(1–301) = 1.8 ± 0.063 μm, n = 11 axons, **, p = 0.004). C and D , the DISC1 Boymaw fusion protein decreases the length of mitochondria (ctrl = 1.81 ± 0.0858 μm, HA Boymaw = 1.54 ± 0.0644 μm, n = 13 axons, *, p = 0.02). Scale bar = 10 μm. E , co-IP experiments from COS7 cells show that DISC1 interacts with Myc Mitofusin1 and -2. F , mitochondrial fractionation from COS7 cells shows that DISC1 is recruited to mitochondria upon overexpression of Myc Mitofusin1 and -2. G , co-IP experiments with rat brain homogenate showing DISC1 to be part of a native complex with Mitofusin1 but not with translocase component TOM20 ( n = 3). IB , immunoblot; IN , input.

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Co-Immunoprecipitation Assay, Fractionation, Over Expression, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: The DISC1-Boymaw fusion protein inhibits mitochondrial fusion. A , HA Boymaw inhibits mitochondrial fusion in neurons. Neurons were transfected with MtDsRed2 and mitochondrially targeted photoactivatable GFP (ctrl) or coexpressing HA Boymaw. Scale bar = 20 μm. B , the change in area of GFP signal after photoactivation is reduced in HA Boymaw-expressing neurons ( n = 17 control and n = 15 Boymaw-expressing neurons, p < 0.05 at 12–90 s post-photoactivation, **, p < 0.01 at 96–570 s post-photoactivation). C , schematic showing mitochondrial fusion after polyethylene glycol-mediated plasma membrane fusion. D and E , HA Boymaw decreases mitochondrial fusion in COS7 cells ( D ) as quantified by colocalization analysis in E ( n = 15 post-fusion cells from three individual preparations, ***, p = 0.0009). Scale bar = 20 μm.

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Transfection, Expressing, Control, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites *

doi: 10.1074/jbc.M115.699447

Figure Lengend Snippet: The DISC1-Boymaw fusion protein decreases the area of ER-mitochondria contacts. A , three-dimensional renderings of mitochondrial network (Su9 GFP ) and ER (ER dsred ) in COS7 cells upon co expression of HA DISC1 or HA Boymaw. Colocalization shows regions of ER-mitochondria interface. Scale bar = 20 μm, 5 μm on zoomed images ( right hand panel ). B , colocalization of HA DISC1 or HA Boymaw with ER-mitochondria contacts. The images of the contact sites were generated from colocalization of images shown in A. C , Manders colocalization coefficient of images in A shows a decrease in area of ER-mitochondria contacts upon HA Boymaw expression ( n = 15 cells from three experiments, ctrl versus Boymaw p < 0.05). NS , not significant. D , Manders coefficient for colocalization of HA DISC1 or HA Boymaw with ER-mitochondria contact sites. HA Boymaw shows higher colocalization than HA DISC1 (*, p = 0.03). E , structured illumination microscopy showing ER and mitochondrial network in SH-SY5Y cells. F , structured illumination microscopy shows partial colocalization between endogenous DISC1 and ER-mitochondria contacts in SH-SY5Y cells. Scale bar = 10 μm (zoom 1 μm). G , line scan showing signal intensities of DISC1 and ER-mitochondria contacts. A.U. = arbitrary units.

Article Snippet: Anti-human DISC1 (14F2) was described previously (WB and immunofluorescence 1:100 mouse) ( ); anti-Rhot1 against Miro was from Atlas Antibodies (HPA010687 for proximity ligation assays and AMAb90854 for immunoprecipitation), and anti-TRAK1 was also from Atlas Antibodies (HPA005853).

Techniques: Expressing, Generated, Microscopy

Journal: Nature medicine

Article Title: Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

doi: 10.1038/nm.3990

Figure Lengend Snippet: Dystrophin regulates PAR polarity protein localization. ( a ) Immunostaining for Dmd C-terminal (green), Dag1 (red), itga7 (cyan) and DAPI (blue) of cultured myofiber at 36 h. ( b ) Quantification of Dmd expression (rod domain) and localization in satellite cells of cultured myofibers of WT mice at 0, 12, 24, and 36 h. Only undivided cells were quantified. n = 3 mice, approx. 50 cells per mice. ( c ) Representative micrograph ( n = 10 micrographs per condition) of proximity ligation assay (PLA) for Dmd (Dy4/6D3 clone) and Mark2 (red) and for ( d ) Dmd and Pard3 (red), along with immunostaining for itga7 (green) and DAPI (blue) on cultured myofibers from WT and mdx mice at 36 h. n = 3 mice. ( e ) Representative micrograph ( n = 10 micrographs per condition) of proximity ligation assay (PLA) between Dag1 and Mark2 (red), and between ( f ) Dag1 and Pard3 (red), along with immunostaining for itga7 (green) and DAPI (blue) on cultured myofibers from WT and mdx mice at 36 h. n = 3 mice. ( g ) Example ( n = 10 micrographs per condition) of polarity protein distribution from immunostaining for Mark2 (green), Pard3 (red), itga7 (white) and DAPI (blue) of cultured myofiber from WT and mdx mice at 36 h. n = 3 mice. ( h , i ) Quantification for ( h ) Mark2 and ( i ) Pard3 expression and localization in satellite cells of cultured myofibers of WT and mdx mice at 36 h. n = 4 mice, 30–50 cells per mice. ( j ) Schematic representation of the polarity establishment in WT and mdx satellite cells. Error bars represent means ± SEM. P * < 0.05, ** P < 0.01, *** P < 0.005. Statistical significance was calculated by Student’s t test. Scale bars, 5 μm.

Article Snippet: We used the following antibodies: mouse anti-Pax7 (DSHB), chicken anti-GFP (cat# ab13970, Abcam), mouse anti-Dmd C-terminal (MANDRA1, clone 7A10, DSHB), rabbit anti-Dmd C-terminal (cat# ab15277, Abcam), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# VP-D508, vector labs), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# DYS1-CE-S, Leica microsystems), mouse anti-Dmd N-terminal (MANEX1011B, clone 1C7, DSHB), mouse anti-Dag1 (cat# ab49515, Abcam), rabbit anti-Dag1 (cat# D1945, Sigma), rabbit anti-Mark2 (cat# 9118S, Cell signaling technology), rabbit anti-phospho-Mark2 AF647-conjugated (cat# bs-5742R, Bioss), rabbit anti-Pard3 (cat# 07-330, Millipore), rabbit anti-phospho-Aurk (cat# 2914S, Cell Signaling technology), rat anti-itga7 AF647-conjugated (clone R2F2, cat# 67-0010-10, AbLab), mouse anti-itga7 (cat# K0046-3, Cedarlane), rat anti-laminin (cat# L0663, Sigma), rabbit anti-Myod1 (C-20, cat# sc-304, Santa Cruz), rabbit anti-Myog (M225, cat# sc-576, Santa Cruz).

Techniques: Immunostaining, Cell Culture, Expressing, Proximity Ligation Assay

Journal: Nature medicine

Article Title: Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

doi: 10.1038/nm.3990

Figure Lengend Snippet: PAR polarity proteins are required for muscle stem cell asymmetric divisions. ( a , b ) Myofibers from Myf5-Cre:R26R-YFP mice cultured for 42 h and immunostained for YFP (green), Pax7 (blue) and Dmd (Dy4/6D3, red; left panel), or Mark2 (red; middle panel), or Pard3 (red; right panel) in ( a ) asymmetric cell pairs (YFP-positive/YFP-negative pairs) and ( b ) symmetric YFP-negative cell pairs (YFP-negative/YFP-negative pairs). Representative pictures from n = 10–20 pictures per condition. ( c , d ) Quantification of asymmetric divisions relative to total satellite stem cell divisions in cultured myofibers of Myf5-Cre:R26R-YFP mice following knockdown of ( c ) Mark2 (si Mark2 ) or ( d ) Pard3 (si Pard3 ) compared to scramble siRNA (siSCR). ( e ) Quantification of Myog-expressing cells per fiber and ( f ) total myogenic cells (Pax7-expressing or Myog-expressing cells) per fiber from WT myofibers cultured 72 h treated with si Mark2 , si Pard3 , or siSCR. ( c – f ) n = 3 mice except for c where n = 5 and d where n = 4, 30–40 myofibers per mice. ( g ) Representative pictures ( n > 10 pictures) from WT and Mark2 −/− section from TA muscle immunostained for laminin (green), Pax7 (red), and DAPI (blue). n = 3 mice. ( h ) Distribution of minimal fiber feret size from the TA muscle of Mark2 −/− mice (blue) and WT littermate (red). n = 3 mice, >300 fibers counted per mouse. ( i ) Representative pictures ( n = 5-10 pictures per condition) of immunostaining for Mark2 (green), Pard3 (red), itga7 (white), and DAPI (blue) of cultured myofiber from WT and Mark2 −/− mice at 36 h. n = 3 mice. Error bars represent means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005. Statistical significance was calculated by Student’s t test. ( a , b , i ) Scale bars, 5 μm and ( g )10 μm.

Article Snippet: We used the following antibodies: mouse anti-Pax7 (DSHB), chicken anti-GFP (cat# ab13970, Abcam), mouse anti-Dmd C-terminal (MANDRA1, clone 7A10, DSHB), rabbit anti-Dmd C-terminal (cat# ab15277, Abcam), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# VP-D508, vector labs), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# DYS1-CE-S, Leica microsystems), mouse anti-Dmd N-terminal (MANEX1011B, clone 1C7, DSHB), mouse anti-Dag1 (cat# ab49515, Abcam), rabbit anti-Dag1 (cat# D1945, Sigma), rabbit anti-Mark2 (cat# 9118S, Cell signaling technology), rabbit anti-phospho-Mark2 AF647-conjugated (cat# bs-5742R, Bioss), rabbit anti-Pard3 (cat# 07-330, Millipore), rabbit anti-phospho-Aurk (cat# 2914S, Cell Signaling technology), rat anti-itga7 AF647-conjugated (clone R2F2, cat# 67-0010-10, AbLab), mouse anti-itga7 (cat# K0046-3, Cedarlane), rat anti-laminin (cat# L0663, Sigma), rabbit anti-Myod1 (C-20, cat# sc-304, Santa Cruz), rabbit anti-Myog (M225, cat# sc-576, Santa Cruz).

Techniques: Cell Culture, Expressing, Immunostaining

Journal: Nature medicine

Article Title: Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

doi: 10.1038/nm.3990

Figure Lengend Snippet: Dystrophin-deficient satellite cells display impaired mitotic spindle orientation and loss of apicobasal division. ( a ) Representative pictures ( n > 50 pictures) of immunostaining for phospho-Aurora kinase (p-Aurk; green), Pax7 (red), and DAPI (blue) of cultured myofibers from WT mice at 36 h. ( b ) Representative images ( n > 20 pictures) of abnormal mitotic events of cultured myofibers from mdx mice at 36 h immunostained for p-Aurk (green), Pax7 (red), and DAPI (blue). ( a , b ) Mitotic centrosomes (arrowheads) are stained with anti-p-Aurk antibody in prophase and metaphase cells. ( c ) Proportion of mitotic satellite cells on cultured myofibers from WT and mdx mice at 36 h. ( d ) Quantification of abnormal, versus planar, and apicobasal orientated mitotic spindles in satellite cells on myofibers from WT and mdx mice after 36 h of culture. ( c , d ) n = 3 WT mice and n = 4 mdx mice, approximately 100 cells total per condition. ( e ) Example of immunostaining ( n = 10 pictures) for Dmd rod domain (green), itga7 (red), p-Aurk (white) and DAPI (blue) in a mitotic satellite cell undergoing an apicobasal division. ( f ) Examples of mitotic satellite cells ( n > 10 pictures) from immunostaining for itga7 (green), p-Aurk (red), and DAPI (blue) of cultured myofiber from Mark2 −/− mice at 36 h. n = 3 mice. ( g ) Quantification of abnormal, versus planar, and apicobasal orientated mitotic spindles in satellite cells on myofibers from WT and Mark2 −/− mice after 36 h of culture. n = 3 mice, >20 fibers counted per condition. ( c , d , g ) Error bars represent means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005. Statistical significance was calculated by Student’s t test. Scale bars, 5 μm.

Article Snippet: We used the following antibodies: mouse anti-Pax7 (DSHB), chicken anti-GFP (cat# ab13970, Abcam), mouse anti-Dmd C-terminal (MANDRA1, clone 7A10, DSHB), rabbit anti-Dmd C-terminal (cat# ab15277, Abcam), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# VP-D508, vector labs), mouse anti-Dmd rod domain (clone Dy4/6D3, cat# DYS1-CE-S, Leica microsystems), mouse anti-Dmd N-terminal (MANEX1011B, clone 1C7, DSHB), mouse anti-Dag1 (cat# ab49515, Abcam), rabbit anti-Dag1 (cat# D1945, Sigma), rabbit anti-Mark2 (cat# 9118S, Cell signaling technology), rabbit anti-phospho-Mark2 AF647-conjugated (cat# bs-5742R, Bioss), rabbit anti-Pard3 (cat# 07-330, Millipore), rabbit anti-phospho-Aurk (cat# 2914S, Cell Signaling technology), rat anti-itga7 AF647-conjugated (clone R2F2, cat# 67-0010-10, AbLab), mouse anti-itga7 (cat# K0046-3, Cedarlane), rat anti-laminin (cat# L0663, Sigma), rabbit anti-Myod1 (C-20, cat# sc-304, Santa Cruz), rabbit anti-Myog (M225, cat# sc-576, Santa Cruz).

Techniques: Immunostaining, Cell Culture, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho isoforms, sequence, and Western blot. A, Structure of the two isoforms of α-Klotho. Isoform 1 represents the full-length protein and contains a signal sequence domain (SS), two homologous domains (KL1, KL2), a short transmembrane domain (TM), and a short cytoplasmic tail. Shown is the site of the epitope for the antibody used in our experiments: AA 800 to 900, KL2. This epitope is absent from Isoform 2, a soluble, secreted protein that arises from alternative RNA splicing and contains only AA 1–549, and where the terminal 15 residues are replaced by the sequence shown. B, The full-length α-Klotho protein sequence of 1012 AA is shown, with KL1 and KL2 shown in red and green respectively, and TM highlighted (black). The peptides giving rise to the PRM signature are also shown (bold typeface; common to isoforms 1 and 2, red; exclusive to full-length α-Klotho, isoform 1, blue). C and D, Western blot analysis of cell lysates (C) and tissues (D) supports the presence of the full-length α-Klotho. Full-length rh α-Klotho protein (rh-α-Klotho). EC, epithelial cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Sequencing, Western Blot

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: α-Klotho protein expression and distribution in human epithelial and reproductive tissues. IHC, positive staining (brown) was found in all the cellular layers of the epidermis (A) and appendage tissue such as hair follicle and sebaceous gland (B). Intestinal expression was primarily found in epithelial cells as illustrated in jejunum (C) and colon (D). In reproductive tissues, positive staining was found in epithelial Sertoli cells (E), testosterone producing Leydig cells (illustrated with white arrows) of the testis (F), and epithelial cells of the prostate gland G). H–K, In mammary tissue (H), endometrium of uterus (I), and endometrium of salpinx (K), the epithelial cell layer was staining strongly for α-Klotho protein; insets are larger magnifications of the epithelial layer. n ≥ 5 for each tissue.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Staining

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Mass spectrometry characterization of the transmembrane α-Klotho protein in human tissues and cells: extracellular α-Klotho peptide GLFYVDFLSQKD (exon 3). A–D, Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho rh full-length α-Klotho protein (rh-α-Klotho) (A), renal proximal tubular epithelial cells (B), kidney tissue (C), and renal artery (D). E–G, The full-length specific (isoform 1) αKlotho peptide LWITMNEPYTR (exon 4). Representative mass spectrometry spectra (left) and Skyline data (right) confirmed the presence of full-length α-Klotho. E), rh full-length α-Klotho protein (rh-α-Klotho); F, kidney tissue; G, renal artery; and H, neuronal cells.

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Mass Spectrometry, Targeted Proteomics

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: α-Klotho Expression in Human Tissues

doi: 10.1210/jc.2015-1800

Figure Lengend Snippet: Confirmation of Transmembrane α-Klotho Protein Expression in Human Tissues and Cells

Article Snippet: Cell lysates were obtained from commercially available primary cell cultures (ScienCell Research Laboratories), including: Human Epidermal Keratinocyte Lysate-adult (product code HEKL-a, catalog no. 2116), Human Prostate Epithelial Cell Lysate (product code HPrEpiCL, catalog no. 4416), Human Mammary Epithelial Cell Lysate (product code HMEpiCL, catalog no. 7616), Human Renal Proximal Tubular Epithelial Cell Lysate (product code HRPTEpiCL, catalog no. 4106), and Human Neuron Lysate (product code HNL, catalog no. 1526).

Techniques: Expressing, Recombinant

Journal: Molecular Cell

Article Title: Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation

doi: 10.1016/j.molcel.2019.06.002

Figure Lengend Snippet: Integration of RAP80 and DUB Specificity of BRCA1-A (A) The position of the UEV-N, RWD, and UEV-C domains of BRE and the VWA domain of MERIT40 (shown as cartoon) within the BRCA1-A complex (shown as surface). RAP80 is embedded into the complex through contacts with BRE, MERIT40, and ABRAXAS. (B) BRCA1-A complex processes (Ub) 4 faster than (Ub) 3 and (Ub) 2 . K63-linked ubiquitin substrate (150 ng/lane) was incubated with 5 nM BRCA1-A on ice and analyzed by silver-stained SDS-PAGE. Under these conditions, (Ub) 4 is cleaved within 5 min, whereas (Ub) 2 and (Ub) 3 are not cleaved within 7 h. (C) Tetra-ubiquitin chains are pre-oriented on BRCA1-A complex during cleavage, as evident from comparison of the degradation of K63-linked (Ub) 4 substrate labeled with TAMRA at the proximal and distal ubiquitin, respectively. Degradation products of 1 μM substrate incubated with 10 nM BRCA1-A at room temperature were resolved on an SDS-PAGE; TAMRA was subsequently visualized.

Article Snippet: K63-(Ub) 4 , TAMRA label on proximal ubiquitin , LifeSensors , Cat# SI6304T.

Techniques: Ubiquitin Proteomics, Incubation, Staining, SDS Page, Comparison, Labeling

Journal: Molecular Cell

Article Title: Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation

doi: 10.1016/j.molcel.2019.06.002

Figure Lengend Snippet: BRCA1-A Forms a Defined High-Affinity Complex with BRCA1 (A) In an MST assay measuring binding of labeled BRCA1-BRCT to full-length BRCA1-A complex including phosphorylated p-Ser404/406 ABRAXAS C terminus, BRCA1-BRCT is bound with nanomolar affinity. The assay measures change of relative fluorescence during heating; values are F norm = F hot / F cold . Error bars represent mean ± SD of n = 4 replicates. Back titration with unlabeled BRCA1-BRCT confirms nanomolar affinity. Error bars represent mean ± SD of n = 4 replicates. (B) Crosslinking network of the BRCA1-A-BRCA1 complex. Proteins are shown schematically as bars. Crosslinks are shown as black lines. The crosslink between K360 of BRE and K1750 of BRCA1 is conditional on the presence of p-Ser404/406 phosphorylation on ABRAXAS C terminus. (C) Model of the BRCA1-A-BRCA1 high-affinity complex. One protomer of ABRAXAS, BRE, and RAP80 is shown as cartoon, while the remainder of the BRCA1-A complex is shown as gray surface. A BRCA1-BRCT dimer (blue, cartoon) is depicted in a position that localizes K1750 of BRCA1 proximal to K360 of BRE. The unstructured C-terminal regions of ABRAXAS are depicted schematically as orange and gray lines. A di-ubiquitin (green, surface) is shown modeled into the active site.

Article Snippet: K63-(Ub) 4 , TAMRA label on proximal ubiquitin , LifeSensors , Cat# SI6304T.

Techniques: Binding Assay, Labeling, Fluorescence, Titration, Phospho-proteomics, Ubiquitin Proteomics

Journal: Molecular Cell

Article Title: Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation

doi: 10.1016/j.molcel.2019.06.002

Figure Lengend Snippet: Metabolic Enzyme SHMT2α Binds Specifically to BRISC, Inactivating It (A) The cryo-EM structure of BRISC-SHMT2α reveals why SHMT2α binds BRISC, but not BRCA1-A. While the ABRO1 isoleucine (Ile133) proximal to SHMT2α is conserved in ABRAXAS, it points away from SHMT2α due to insertion of a proline (Pro137) into ABRAXAS. (B) SHMT2 clashes with the position of the proximal ubiquitin of a ubiquitin dimer bound to the active site. Modeling di-ubiquitin (red, surface) into the BRCC36 active site reveals a substantial clash with the position of SHMT2α (green) in the BRISC-SHMT2α map (semitransparent surface). (C) SHMT2α is a potent inhibitor of BRISC (2 nM), but not of BRCA1-A (5 nM), as shown by SHMT2α-mediated inactivation of BRISC in a DUB-activity assay using IQF K63-linked di-Ubiquitin. A BRISC-RAP80 fusion construct (2 nM) containing the MPN domain of ABRO1 combined with the C terminus of ABRAXAS integrates RAP80 like BRCA1-A but is inhibited by SHMT2α like BRISC. Error bars represent mean ± SD of n = 3 replicates.

Article Snippet: K63-(Ub) 4 , TAMRA label on proximal ubiquitin , LifeSensors , Cat# SI6304T.

Techniques: Cryo-EM Sample Prep, Ubiquitin Proteomics, Activity Assay, Construct

Journal: Molecular Cell

Article Title: Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation

doi: 10.1016/j.molcel.2019.06.002

Figure Lengend Snippet:

Article Snippet: K63-(Ub) 4 , TAMRA label on proximal ubiquitin , LifeSensors , Cat# SI6304T.

Techniques: Virus, Recombinant, Protease Inhibitor, Ubiquitin Proteomics, Expressing, FLAG-tag, Software