Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: Skeletal muscle p53 expression under metabolic stress and generation of an inducible skeletal muscle-specific p53 knockout model. (A–B) p53 protein expression in the gastrocnemius muscle of male mice fed a high-fat diet (HFD) for 12 weeks ( n = 6 per group). (C–D) p53 protein expression in the deltoid muscle of patients with type 2 diabetes and non-diabetic individuals ( n = 5-7 per group). (E) Schematic diagram illustrating the generation of the inducible skeletal muscle-specific p53 knockout (iMp53 KO) mouse model. (F) Experimental timeline depicting doxycycline administration and dietary intervention. GAPDH was used as a loading control. All samples were biologically independent. Data are presented as mean ± SE. Statistical analysis was performed using a two-tailed Student's t -test for comparisons between two groups. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Abbreviations: Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Expressing, Knock-Out, Control, Two Tailed Test

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency does not alter insulin sensitivity under metabolically normal conditions. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing chow diet for 19 weeks. (A) p53 gene expression in the gastrocnemius muscle ( n = 5 per group). (B) Average daily calorie intake ( n = 3 per group). (C) Accumulated food intake during the experimental period ( n = 3 per group). (D) Initial baseline body weight, final body weight, and weight gain during the experimental period ( n = 9–10 per group). (E) Weights of skeletal muscle, epididymal fat tissue, and liver ( n = 4 per group). (F–G) Blood glucose levels and area under the curve (AUC) during the glucose tolerance test ( n = 9–10 per group). (H–I) Plasma insulin levels and AUC during the glucose tolerance test ( n = 9–10 per group). (J–K) Percent reduction in blood glucose from baseline and AUC during the insulin tolerance test ( n = 9–10 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t-test for comparisons between two groups. ∗∗∗∗ p < 0.0001. Abbreviations: BW, body weight; Con, control; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Metabolic Labelling, Control, Gene Expression, Clinical Proteomics, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency improves insulin resistance in HFD-fed obese mice. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Data shown in panels H–K were collected after 10 weeks of HFD feeding, while data in panels L and M were obtained after 19 weeks of HFD feeding. (A) p53 gene expression in the gastrocnemius muscle ( n = 5 per group). (B–C) p53 protein expression in the gastrocnemius muscle ( n = 7 per group). (D) Average daily calorie intake ( n = 3 per group). (E) Accumulated food intake during the experimental period ( n = 3 per group). (F) Initial baseline body weight, final body weight, and weight gain during the experimental period ( n = 7 per group). (G) Weights of skeletal muscle, epididymal fat tissue, and liver ( n = 7 per group). (H–I) Blood glucose levels and area under the curve (AUC) during the glucose tolerance test ( n = 7 per group). (J–K) Plasma insulin levels and AUC during the glucose tolerance test ( n = 7 per group). (L–M) Percent reduction in blood glucose from baseline and AUC during the insulin tolerance test ( n = 7 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was assessed using a two-tailed Student's t -test for comparisons between two groups. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Abbreviations: BW, body weight; Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Control, Gene Expression, Expressing, Clinical Proteomics, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency enhances insulin-stimulated glucose disposal and signaling in skeletal muscle of HFD-fed obese mice. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Following HFD feeding, a hyperinsulinemic-euglycemic clamp test was performed to assess whole-body and tissue-specific insulin sensitivity (A–C; n = 7 per group), including whole-body glucose turnover (A), glucose uptake in the soleus muscle (B), and hepatic glucose production (C). To evaluate insulin signaling, mice were fasted overnight and administered insulin (1.5 U/kg) intraperitoneally; gastrocnemius muscles were harvested 10 min post-injection, and insulin signaling proteins were analyzed by Western blot (D–J; n = 7 per group). GAPDH was used as a loading control, and phosphorylated proteins were normalized to their respective total protein levels. Representative blots of insulin signaling proteins (D), phosphorylated Akt (pAkt; Ser473) levels (E), total Akt (F), phosphorylated AS160 (pAS160) levels (G), total AS160 (H), phosphorylated GSK3β (pGSK3β) levels (I), and total GSK3β (J). All samples were biologically independent. Data are presented as mean ± SE, and statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Abbreviations: AS160, Akt substrate of 160 kDa; Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3β, glycogen synthase kinase 3β; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Control, Muscles, Injection, Western Blot, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency improves mitochondrial membrane potential and respiratory capacity. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Protein levels of mitochondrial electron transport chain complexes and PGC1α in the gastrocnemius muscle were analyzed by Western blot (A–F; n = 7 per group), with GAPDH used as a loading control. Representative Western blots (A) quantification of PGC1α (B) and quantification of mitochondrial electron transport chain complex proteins (C–F). In vitro , C2C12 myoblasts were transfected with shCon or shp53, induced to differentiate, and then treated with 0.25 mM palmitic acid for 12 h (G–J). Differentiated myotubes were incubated with 10 μM JC-1 dye for 1 h; fluorescence images were captured using a fluorescence microscope, and signal intensities were quantified using a fluorescence microplate reader (G–H). Mitochondrial membrane potential was assessed by JC-1 fluorescence; representative images are shown (G) and quantification analysis of JC-1 signal (H; n = 3 per group). Mitochondrial oxygen consumption rate in C2C12 myotubes was measured using the Seahorse system (I–J; n = 3 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups and one-way ANOVA followed by Tukey's post hoc test for comparisons among four groups. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Abbreviations: Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, inducible skeletal muscle-specific p53 knockout; PA, palmitic acid; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1 alpha; shCon, control shRNA-transfected C2C12 cells; shp53, p53 shRNA-transfected C2C12 cells.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Membrane, Control, Western Blot, In Vitro, Transfection, Incubation, Fluorescence, Microscopy, Two Tailed Test, Knock-Out, shRNA

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency reduces the area of mitochondria-associated membranes in skeletal muscle of HFD-fed obese mice. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Transmission electron microscopy was used to visualize mitochondria in the tibialis anterior muscle at magnifications of ×15,000 and × 60,000 (inset) (A). Mitochondrial density (number per μm 2 ; B) and mitochondrial area (C) were quantified (B–C; n = 3 per group). The ratio of mitochondria-associated membrane (MAM) length relative to mitochondrial perimeter was also quantified (D; n = 5 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups. ∗∗ p < 0.01. Abbreviations: Con, control; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Control, Transmission Assay, Electron Microscopy, Membrane, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: Skeletal muscle p53 interacts with mitochondria-associated membrane (MAM) components and regulates mitochondrial calcium loading. Eight-week-old male mice were fed either a chow diet or a high-fat diet (HFD) for 12 weeks, after which expression levels of MAM-related proteins and SERCA1 in the tibialis anterior muscle were analyzed by Western blot (A–B; n = 6 per group). Eight-week-old control and iMp53 KO male mice were then fed a doxycycline-containing HFD for 19 weeks, and expression levels of MAM components and SERCA1 were again assessed in the gastrocnemius muscle via Western blot (C–D; n = 7 per group). GAPDH was used as a loading control. GRP75-targeted immunoprecipitation was performed using gastrocnemius muscle lysates from control and p53-deficient mice to assess protein–protein interactions (E–F), and p53-targeted immunoprecipitation was conducted to evaluate its interaction with MAM components (G–H; n = 3 per group). To assess mitochondrial calcium dynamics, C2C12 cells transfected with either shCon or shp53 were treated with 0.25 mM palmitic acid for 1 h or 3 h on day 5 of differentiation. Mitochondrial calcium levels were visualized via confocal microscopy (I) and quantified by flow cytometry (J; n = 6 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t- test for comparisons between two groups and one-way ANOVA followed by Tukey's post hoc test for comparisons among four or more groups. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Abbreviations: Con, control; KO, inducible skeletal muscle-specific p53 knockout; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP75, glucose-regulated protein 75; IP3R, inositol 1,4,5-trisphosphate receptor; PA, palmitic acid; SERCA1, sarcoplasmic reticulum Ca 2+ ATPase 1; shCon, control shRNA-transfected C2C12 cell; shp53, p53 shRNA-transfected C2C12 cell; VDAC, voltage-dependent anion channel.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Membrane, Expressing, Western Blot, Control, Immunoprecipitation, Protein-Protein interactions, Transfection, Confocal Microscopy, Flow Cytometry, Two Tailed Test, Knock-Out, shRNA

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 protein expression is positively correlated with mitochondria-associated membrane (MAM)-related proteins in human skeletal muscle. Protein expression levels of IP3R, GRP75, VDAC, and SERCA1 were analyzed in the deltoid muscle samples from non-diabetic and diabetic individuals (A–D; n = 5-7 per group), with GAPDH used as a loading control. Correlations between p53 protein levels and the expression of MAM-related proteins were assessed across all human skeletal muscle samples (E–H; n = 24). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP75, glucose-regulated protein 75; IP3R, inositol 1,4,5-trisphosphate receptor; SERCA1, sarcoplasmic reticulum Ca 2+ -ATPase 1; VDAC, voltage-dependent anion channel.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Expressing, Membrane, Control, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 1. p53 and WOX1 binding interactions. A, Molt-4 T cells were exposed to UV light (240 mJ/cm2), followed by culturing for 10–120 min. Both cytosolic and nuclear fractions were pre- pared and examined for phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (p-WOX1) and their nuclear translocation. UV light also increased the levels of nuclear histone H3 and its acetylation at lysine 9 (H3k9). By immunoprecipitation (IP) using p53 IgG antibody, UV light was shown to induce binding of cytosolic p53 with WOX1 in Molt-4 T cells.B,weconfirmedtheUVlight-inducednuclear translocation of p53 and WOX1 in Molt-4 cells by immunofluorescence microscopy. Cells were stained with IgG antibodies against WWOX (N19; goat) and full-length p53 (FL-393; rabbit), respec- tively (3, 4). Secondary staining was performed using anti-goat IgG (conjugated with Texas Red) and anti-rabbit IgG (conjugated with Cy2). Nuclei were stained with 4,6-diamidino-2-phenylindole. C, monocytic U937 cells were exposed to UV light (240 mJ/cm2) and shown to have increased phos- phorylation of cytosolic p53 at Ser46 and WOX1 at Tyr33, and p53 binding with p-WOX1. D, similarly, neuroblastoma SK-N-SH cells were exposed to deferroxamine (500 M) for 1 h to induce hypoxic stress and shown to have an increased binding of p53 with WOX1.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Binding Assay, Phospho-proteomics, Translocation Assay, Immunoprecipitation, Immunofluorescence, Microscopy, Staining

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 3. Tyr33-phosphorylated WOX1 binds Ser46-phosphorylated p53 but not Ser46-deleted p53. p53-negative H1299 cells were transfected with DsRed-tagged wild type p53, p53S46, or DsRed alone by electroporation. The cells were cultured for 48 h and examined by fluorescence microscopy. More than 70% of cells expressed DsRed. UV light increased the binding of p-WOX1 with the wild type p53-DsRed, but not p53S46- DsRed, as determined by co-immunoprecipitation using antibodies against p-WOX1 (25). The ectopically expressed proteins are shown (see the bottom panel). IgH, IgG heavy chain.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Transfection, Electroporation, Cell Culture, Fluorescence, Microscopy, Binding Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 2. Mapping of p53 and WOX1 interac- tions by yeast two-hybrid analysis and temper- ature-dependent WOX1 phosphorylation. A, analysis of protein/protein interaction in the cyto- plasm was performed using a Ras rescue-based yeast two-hybrid system (3, 10, 25, 29). In positive controls, binding of WOX1 with p53 and MafB self- interactionareshown,asevidencedbythegrowth of yeast at 37 °C using selective agarose plates containing galactose. In negative controls, no yeast growth at 37 °C was observed for the empty pSos/pMyr vectors and collagenase and lamin C. Human p53 physically interacted with the N-ter- minal WW domains of murine WOX1. Deletion of an N-terminal proline-rich segment (amino acids 66–100; see block No. 2) from the full-length p53 did not abrogate the binding. Similarly, deletion of Thr18 or Ser20 in p53 could not prevent its binding with WOX1. In contrast, deletion of Ser46 in p53 significantly reduced the binding. Alterations of Tyr33 to Arg in the first WW domain of WOX1 abol- ished its binding with p53. p53 structure was as follows: transcriptional activation domain (1); pro- line-rich region (2); DNA-binding domain (3); tet- ramerization domain (4). B, to examine WOX1 phos- phorylation at Tyr33, some yeast cells were grown at room temperature for 2 days in a galactose-contain- ing broth (to reach a preplateau stage), followed by culturing at 37 °C for 1 h to induce the activation of Ras pathway. Heat induced the expression of p53, WOX1, and its phosphorylation at Tyr33 (p-WOX1) in yeastcellstransfectedwithwildtypep53andWOX1. Incontrast,noWOX1phosphorylationwasobserved in cells transfected with p53S46 mutant and wild type WOX1.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Phospho-proteomics, Binding Assay, Blocking Assay, Activation Assay, Expressing, Transfection, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

doi: 10.1074/jbc.m505590200

Figure Lengend Snippet: FIGURE 10. A schematic model for p53 interactions with WOX1 and MDM2. A, UV light induces phosphorylation of p53 at Ser46 and WOX1 at Tyr33 and their complex formation in the presence of MDM2. Nutlin-3 inhibits MDM2 and appears to alter p53 conformation (p53*), thereby stabilizing p53 and its interaction with WOX1. This p53 is not phosphorylated at key serines. UV light restores the complex formation of p-p53-p- WOX1-MDM2. B, WOX1 alone binds MDM2 probably via its C-terminal short-chain alco- hol dehydrogenase/reductase domain, and nutlin-3 dissociates the binding. UV light restores the binding.

Article Snippet: Additional specific antibodies used in Western blotting were against the following proteins: polyclonal IgG against WWOX (N-19), MDM2 (C-18), and p53 (FL-393) and monoclonal IgG against p53 (Pab240) from Santa Cruz Biotechnology; p53 and I B from BD Biosciences; phospho-p53 at Ser15 from Calbiochem; phospho-p53 at Ser46 from R&D Systems; -tubulin from Accurate Chemicals; and histones and their acetylated forms from Cell Signaling. cDNA Expression Constructs, Transfection and Expression in Cell Lines, and Stable Transfectants—The following expression constructs were made as previously described: 1) murine EGFP-WOX1 (3), 2) murine dominant negativeWOX1 (dn-WOX1) tagged with EGFP (25), 3) human wild type p53 tagged with DsRed (p53-pDsRedN1) (29), and 4) human p53-pDsRedN1 with Ser46 deletion (29).

Techniques: Phospho-proteomics, Binding Assay

Journal: Biological chemistry

Article Title: Lysine acetyltransferase Tip60 acetylates the APP adaptor Fe65 to increase its transcriptional activity.

doi: 10.1515/hsz-2020-0279

Figure Lengend Snippet: Figure 5: Effect of Sirt1 and different KATs on Fe65 acetylation levels. (A–C) Dynabeads-Streptavidin-based pulldown of SBP-myc-Fe65 from HEK293 cells co-transfected with myc-Tip60 and either wt or mutant (H363Y) FLAG-Sirt1, followed by subsequent Western blotting of lysates (L) and eluates (E). (A) Representative western blots. Band marked by a star is unspecific staining by the anti-FLAG antibody. (B) Quantification of eluates showing the ratio of acetylated Fe65 vs. Fe65 and acetylated Tip60 vs. Tip60. (C) Quantification of co- precipitated Tip60. Mean ± SEM of n = 4 are shown for (B) and (C) (two-tailed t-test). (D) SBP-myc-Fe65 or SBP-myc-p53 were co-expressed with wt myc-Tip60, ΔKAT myc-Tip60 or wt hMOF-myc in HEK293 cells, followed by pulldown and Western blot analysis. In contrast to wt Tip60, its closest homolog hMOF does not acetylate Fe65. Acetylation of SBP-myc-p53 through wt myc-Tip60 and hMOF-myc confirms the activity of hMOF-myc. (E) Confocal microscopy images of hMOF-myc show a uniform nuclear localization (upper row). Co-transfection of Cer-Fe65 with hMOF-myc did not result in Fe65 localizing to specific nuclear structures (lower row). Scale bar: 10 µm. (F) SBP-myc-Fe65 was co-expressed with Cit-3HA, myc-Tip60, GFP-GCN5 or CBP-HA in HEK293 cells, followed by pulldown and Western blot

Article Snippet: GFP-p53 (#12091, Addgene, deposited by Tyler Jacks) (Boyd et al. 2000) was used to PCR amplify p53, which was fused to a N-terminal SBP-myc tag and cloned into the pUKBK vector system (Kohli et al. 2012) to create SBP-myc-p53.

Techniques: Transfection, Mutagenesis, Western Blot, Staining, Two Tailed Test, Activity Assay, Confocal Microscopy, Cotransfection