Journal: International Journal of Molecular Sciences

Article Title: A Barth Syndrome Patient-Derived D75H Point Mutation in TAFAZZIN Drives Progressive Cardiomyopathy in Mice

doi: 10.3390/ijms25158201

Figure Lengend Snippet: PCR primers used.

Article Snippet: Following blocking in Blotting-Grade Blocker (Bio-Rad) blots were probed with the following antibodies: Acaa2 #237540, Arginase #96183, Bcl2 #16904, Bcl2-interacting protein-3 #109362, Catalase #209211, Cathepsin D #75852, CDKN2A/p16INK4a #211542, Connective tissue growth factor #209780, Galectin-3 #76245, Hadh #154088, Lactate dehydrogenase #52488, nuclear Lamin B1 #229025, Myoglobin #77232, Phospholamban #219626, Phospholamban (phospho S16) #15000, PKA alpha/beta/gamma (phospho T197) #75991, p21 #188224, p53 (phospho S15) #278683, p53 (acetyl K370) #183544, Serca2 #3625, Sequestosome-1 #109012, Sirt3 #246522, Sod2 #68155, TIGAR #62533, Ucp3 #193470; Slc38a1 (Bioss Antibodies #bs-19825R); MDM2 (BioRad #AHP1329); AMPK #2532, Caspase-3 #9662, cleaved Caspase-3 #9661, Cycs #4272, pCaMKII (phospho T286) #12716, Mcu #14997 (Cell Signaling); TP53INP2 (LSBio #LS-C413392); Atp5a1 (Proteintech #14676-1-AP); Acsl1 #PA5-17136, 4-HNE #MA5-27570, LC3B #PA1-46286, VDAC #PA1-954A (Invitrogen); Pla2g6 (Novus Biologicals #NBP1-81586); Ly-6G #sc-53515, p53 #sc-71820, Pten #sc-7974, Pink1 #sc-517353, Taz #sc-365810 (Santa Cruz), GAPDH #G8795, cytosolic Tubulin #T5168 (Sigma).

Techniques:

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: Dissociation constants K d of MDM2 homodimer and MDM2-MDMX heterodimer variants for UbcH5B–Ub.

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques:

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: a Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2-419–C and its S429E substitution using fluorescently labeled Ub and visualized with an Odyssey CLx Imaging System. b Plot of relative ubiquitination activity corresponding to a . c Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2-419–C-His-MDMX-418–C and its MDM2-S429E substitution using fluorescently labeled Ub and visualized with an Odyssey CLx Imaging System. d Plot of relative ubiquitination activity corresponding to c . For b , d , data are presented as mean value ± SD from three independent experiments ( n = 3). e Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2-419–C and its S429E substitution over the indicated times using fluorescently labeled Ub and visualized with an Odyssey CLx Imaging System. f A plot showing the rates of ubiquitination catalyzed by the forms of MDM2 assessed in e . The line represents the regression line from three independent experiments ( n = 3). Asterisks in a , c , and e indicate non-reducible E1–Ub product. Uncropped gel images and InstantBlue-stained gels are shown in Supplementary Fig. .

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques: SDS Page, Labeling, Imaging, Activity Assay, Staining

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: a Cartoon representation of the complex. One MDM2 R is colored green and the other is in magenta. UbcH5B is in cyan and Ub is in yellow. UbcH5B–Ub linkage is indicated. Top and bottom panels are related by 90° rotation about the x -axis. b Surface representation of the complex, colored and oriented as in a (top panel). The N-terminal region preceding the RING domain and the C-terminal tail of MDM2 are indicated. c A cartoon representation of the structure of MDM2-MDMX RING domain heterodimer bound to UbcH5B–Ub (PDB ID: 5MNJ) oriented in the same view as in a (bottom panel). MDM2 R , UbcH5B, and Ub are colored as in a and MDMX RING domain (MDMX R ) is in orange.

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques:

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: Data collection and refinement statistics.

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques:

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: a Close-up view of MDM2 R -UbcH5B interactions. b Close-up view of MDM2 R -Ub and Ub–UbcH5B interactions. c Close-up view of MDM2’s C-terminal tail. A transparent surface representation is shown. d Close-up view of the N-terminal region preceding the MDM2 RING domain. e Close-up view of the N-terminal region preceding the RING domain in the structure of the MDM2-MDMX-UbcH5B–Ub complex (PDB ID: 5MNJ) shown in the same view as in d . a – e are colored as in Fig. . Key residues are shown as sticks. Carbon atoms are colored according to the parent subunit. Nitrogen, oxygen, and sulfur atoms are in blue, red, and gold, respectively. Zinc atoms are depicted as gray spheres. A dashed line indicates hydrogen bonds. f Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2-419–C and variants using fluorescently labeled Ub and visualized with an Odyssey CLx Imaging System. Uncropped gel images and InstantBlue-stained gels are shown in Supplementary Fig. . g Plot of relative ubiquitination activity of MDM2 variants in f . Data are presented as mean value ± SD from three independent experiments ( n = 3).

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques: SDS Page, Labeling, Imaging, Staining, Activity Assay

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: a Sequence alignment of MDM2 RING domain from human and cat. S429 is colored red. The four non-identical residues between human and cat are highlighted in bold. b Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-cat-MDM2-422–C and its S429E substitution using fluorescently labeled Ub and visualized with an Odyssey CLx Imaging System. c Plot of relative ubiquitination activity of cat MDM2 variants in b . d Cartoon representation of the structure of cat MDM2-422–C-pS429 bound to UbcH5B–Ub shown in the same colors and orientation as in Fig. (top panel). pS429 is indicated. e Close-up view of pSer429 in d with polder density map (blue) contoured at 1 σ . f Close-up view of pS429-Ub interactions in d . g Close-up view of S429E in cat MDM2-422–C-S429E-UbcH5B–Ub structure (Supplementary Fig. ) with polder density map (blue) contoured at 1 σ . h Close-up view of S429E-Ub interactions as in g . For e – h , key residues are shown as sticks and colored as in Fig. . Phosphorus atoms are in orange. The water molecule is depicted as a red sphere. Hydrogen bonds are shown as dashed lines and the distances are indicated. i , k , m Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2-419–C variants using indicated fluorescently labeled Ub variants visualized with an Odyssey CLx Imaging System. j , l , n Plots showing the relative ubiquitination activity of MDM2 variants in i , k , m . In c , j , l , n , data are presented as mean value ± SD from three independent experiments ( n = 3). All uncropped gel images and InstantBlue-stained gels are shown in Supplementary Fig. .

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques: Sequencing, SDS Page, Labeling, Imaging, Activity Assay, Staining

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: a Cartoon representation of the structure of cat MDM2-422–C-S429E. b Cartoon representation of the MDM2-MDMX portion of MDM2-MDMX-UbcH5B–Ub complex structure (PDB ID: 5MNJ; left panel) and MDM2-MDMX RING domain structure (PDB ID: 2VJF; right panel) shown in the same view as in a . c Comparison of the location of pS429 in cat MDM2-422–C-pS429-UbcH5B–Ub structure and S429 in MDM2-MDMX-UbcH5B–Ub structure (PDB ID: 5MNJ). The MDM2-MDMX RING domain from the structure of MDM2-MDMX-UbcH5B–Ub complex was superimposed onto the structure of cat MDM2-422–C-pS429-UbcH5B–Ub complex. d Comparison of the location of pS429 in cat MDM2-422–C-pS429-UbcH5B–Ub structure and S429 in MDM2-MDMX RING domain structure (PDB ID: 2VJF). The MDM2-MDMX structure was superimposed onto the structure of cat MDM2-422–C-pS429-UbcH5B–Ub complex. e Close-up views of dimer interaction at the N terminus of MDM2 in the structures of cat MDM2-422–C-pS429-UbcH5B–Ub complex (left panel) and MDM2-MDMX RING domain (PDB ID: 2VJF; right panel). For a – e , all coloring is as described in Fig. , except MDM2 R in the heterodimer is colored gray. Key residues in c – e are shown in sticks and atoms are colored as in Fig. . Distances between the Cα atoms of pS429 and S429 are indicated in the homodimer in c and in the heterodimer in d . f , h , j Reduced SDS-PAGE showing autoubiquitination reactions catalyzed by GST-MDM2-419–C variants using fluorescently labeled Ub and visualized with an Odyssey CLx Imaging System. Asterisks indicate non-reducible E1–Ub product. In f , Ala_ins indicates insertion of an alanine between residues 429 and 430 in MDM2. Uncropped gel images and InstantBlue-stained gels are shown in Supplementary Fig. . g , i , k Plots showing the relative ubiquitination activity of MDM2 variants in f , h , j , respectively. Data are presented as mean value ± SD from three independent experiments ( n = 3).

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques: SDS Page, Labeling, Imaging, Staining, Activity Assay

Journal: Nature Communications

Article Title: Structural basis for DNA damage-induced phosphoregulation of MDM2 RING domain

doi: 10.1038/s41467-020-15783-y

Figure Lengend Snippet: a Immunoblots showing the stability of MDM2 variants from lysates of U2OS mod cells expressing GFP-MDM2 variants treated with cycloheximide for indicated times. The immunoblots were analyzed by anti-GFP or anti-actin antibodies as indicated. b Immunoblots of MDM2 ubiquitination from lysates of U2OS mod cells transfected with plasmids expressing GFP-MDM2 variants or empty vector (EV) along with His-Ub and treated with MG132. The cell lysates and Ni-NTA pull-down products were analyzed by immunoblotting with anti-GFP or anti-actin antibodies as indicated. c Immunoblots showing the effects of MDM2 variants on p53 and p21. Unmodified U2OS cells were transfected with plasmids expressing GFP-MDM2 variants or EV and Myc-tagged p53. Lysates were analyzed by immunoblotting using anti-GFP, anti-Myc tag, anti-p21, or anti-actin antibodies as indicated. d Immunoblots showing the stability of MDM2 variants from lysates of U2OS mod cells expressing GFP-MDM2 variants left untreated (top panel) or treated with etoposide (bottom panel) for 6 h, followed by cycloheximide treatment for indicated times. The immunoblots were analyzed by anti-GFP or anti-actin antibodies as indicated. e Immunoblots showing MDM2 S429 phosphorylation in the absence and presence of etoposide treatment. U2OS mod cells were transfected with plasmids expressing GFP-MDM2 variants or EV and treated with etoposide where indicated. The cell lysates and GFP-Trap pull-down products were analyzed by immunoblotting with anti-GFP, anti-MDM2-pS429, or anti-actin antibodies as indicated. Low = low exposure; high = high exposure. f Immunoblots of MDM2 ubiquitination from lysates of U2OS mod cells transfected with plasmids expressing GFP-MDM2 variants or EV, along with His-Ub in the absence and presence of etoposide. Prior to harvesting, cells were treated with MG132. The cell lysates and Ni-NTA pull-down products were analyzed by immunoblotting with anti-GFP or anti-actin antibodies as indicated. Actin loading control blots were included for all panels. All the experiments were performed in triplicate with similar results. Raw data are provided in Supplementary Fig. .

Article Snippet: The primary antibodies used in this study include mouse anti-GFP (Santa Cruz Biotechnology, cat. no. sc-81045, 1:1000 for Western blot), customized rabbit anti-MDM2-pS429 (Eurogentec, 1:1000 for Western blot), mouse anti-Myc tag (Cell Signaling Technology, cat. no. 2276, 1:1000 for Western blot), rabbit anti-p21 (Cell Signaling Technology, cat. no. 2947, 1:1000 for Western blot), and goat anti-actin (Santa Cruz Biotechnology, cat. no. sc-1616, 1:1000 for Western blot).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: Increased Δ133p53 mRNA in lung carcinoma corresponds with reduction of p21 expression

doi: 10.3892/mmr.2017.6162

Figure Lengend Snippet: mRNA expression of p53 , full-length and Δ133p53, MDM-2 and p21 relative to β- actin (reference gene) for each individual lung carcinoma and corresponding adjacent non-cancerous specimens. The C q values were analyzed by the 2 −ΔΔCq method using Pfaffl analysis. ▪▪▪, 95% confidence interval limit. MDM-2, mouse double minute 2 homolog.

Article Snippet: The rabbit anti-MDM-2 antibody (S166; AP1253e) was purchased from Abgent (San Diego, CA, USA) and used at a 1:1,000 working dilution.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Increased Δ133p53 mRNA in lung carcinoma corresponds with reduction of p21 expression

doi: 10.3892/mmr.2017.6162

Figure Lengend Snippet: Expression of p53 , full-length and Δ133p53, MDM-2 and p21 transcripts in cancerous vs. non-cancerous tissue in (A) non-smokers (B) intermediate smokers (40–70 packs/year) and (C) heavy smokers (140–200 packs/year) using the Rest statistical analytical tool. ▬, difference limit for the relative expression levels. MDM-2, mouse double minute 2 homolog.

Article Snippet: The rabbit anti-MDM-2 antibody (S166; AP1253e) was purchased from Abgent (San Diego, CA, USA) and used at a 1:1,000 working dilution.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Stabilization of HIF-1α and HIF-2α, up-regulation of MYCC and accumulation of stabilized p53 constitute hallmarks of CNS-PNET animal model

doi: 10.1371/journal.pone.0173106

Figure Lengend Snippet: A. TP53 40x, insert- 5X digital (LCAS-R) B: TP53 40x, insert- 5X digital (LCAS-R-12 weeks post-injection) C: TP53 40x, insert- 5X digital (LC26-R-12 weeks post-injection) D: YB-1 40X, insert- 5X digital (LCAS-R-12 weeks post-injection) E: MDM2 5x, F: MDM2 40x, (LCAS-R-12 weeks post-injection) TU-tumor; P-parenchyma; V-ventricle.

Article Snippet: At least two slides (5μm thick) from each FFPE tumor sample were used for the analysis of each antibody presented in this study using standard immunohistochemical methods (see ).The immunohistochemical panel comprised the following antibodies: Anti-Ki-67 (RM-9106, Rabbit monoclonal, 1:200, Thermo scientific), POU5F1/OCT3/4 (LS-B85, Rabbit polyclonal, 1:300, LSBio), Anti-Nestin (ab105389, Rabbit monoclonal, 1:30, abcam), Anti- Sox2 (ab97959, Rabbit polyclonal, 1:200, abcam), Anti-Vimentin (NBP1-97671, Mouse monoclonal, 1:500, Novus Biologicals), Anti-c-MYC (ab32072, Rabbit monoclonal[Y69], 1:500, abcam), Anti-c-MYC-(Phospho S62) (ab185656, Rabbit monoclonal, 1:500, abcam), Anti-MAX (ab101271, Rabbit polyclonal, 1:1000, abcam), Anti-HIF-1α (ab82832, Rabbit polyclonal, 1:100, abcam), Anti-HIF-2α (ab73895, Rabbit polyclonal, 1:250, abcam), Anti-p53 (LS-B7722, Rabbit polyclonal, 1:200, LSBio), Anti-YB1 (ab12148, Rabbit polyclonal, 1:750, abcam), Anti-MDM2 (LS-C199239, Rabbit polyclonal, 1:100, LS Bio).

Techniques: Injection