Journal: iScience

Article Title: SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer

doi: 10.1016/j.isci.2022.105183

Figure Lengend Snippet: WNT5A may link SEL1L-HRD1 ERAD to hepatic proliferation (A and B) Volcano plot (A) and tabular form (B) of proteomics data obtained via TMT-LC/MS analysis of hepatic ER isolated from 2- to 3-months-old mice (n = 3 per group). Lines mark p = value 0.05 (horizontal) or 2-fold changes (vertical). (C–F) Western blot analysis of ER and non-ER fractions from livers of 2-month-old mice (n = 3 per group) (C, black and red arrows mark WNT5A mobility shift), hepatocellular carcinoma and normal liver tissue from the DEN-HFD liver cancer model (n = 2 per group, 2 independent repeats) (D), livers of 10-week-old mice 48-h post partial hepatectomy (n = 3 per group) (E), livers from chronic CCL4 treatment (n = 2 per group) (F). (G) Western blot analysis normal liver and tumor tissues from the year-long-HFD experiment (n = 3 per group). (H and I) Hepatic cDNA microarray analyses of 9-week-old Sel1L Alb livers compared to Sel1L f/f livers: (H) KEGG pathway for Gene Set Enrichment Analysis (GSEA) from differentially expressed hepatic genes with p < 0.05 and fold change >1.29, and (I) Heatmap of proliferation-associated genes as a logarithm of fold change in Sel1L Alb and Hrd1 Alb livers as compared to Sel1L f/f and Hrd1 f/f livers, respectively (n = 3 per group). (J) Western blot analysis of HepG2 and Huh7 cells treated with 500 ng/mL recombinant WNT5A protein for 30 h (2 independent repeats). (K) Western blot analysis of Sel1L f/f and Sel1L Alb livers 2 weeks after i.v. injection of AAV8 expressing shRNA targeting Wnt5A or luciferase control (n = 2–3 per group). Quantitation shown below each blot. Calnexin, ER fraction control; HSP90, loading and non-ER controls. Values, mean ± SEM; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by Student’s t test.

Article Snippet: The following primary antibodies were used: KI67 (1:200, Abcam ab16667); CYCLIN D1 (1:100 for staining, 1:2000 for Western blot, Abcam ab16663); c-MYC (1:1000, MilliporeSigma C3956); V5 (1:5000 for Western blot, 1:250 for immunoprecipitation, Invitrogen R960-25); HA (1:3,000, Sigma H9658); WNT5A (1:2000 for Western blot, 1:100 for immunoprecipitation, Proteintech 55184-1-AP); WNT5B (1:1000, Abclonal A8313); WNT1 (1:1000, Proteintech 27935-1-AP); E-CADHERIN (1:1000, BD 610181); α-TUBULIN (1:2,000, Santa Cruz sc-5286); HSP90 (1:1,000, Abcam ab13492); CALNEXIN (1:5000, Enzo ADI-SPA-860-F); SEL1L (1:2,000, Abcam ab78298); HRD1 (1:300, kindly gifted by Dr. Richard Wojcikiewicz, SUNY Upstate Medical University for Western blot; 1:1000, Sigma HPA024300 for immunostaining of human liver tumors).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Western Blot, Mobility Shift, Microarray, Recombinant, Injection, Expressing, shRNA, Luciferase, Quantitation Assay

Journal: iScience

Article Title: SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer

doi: 10.1016/j.isci.2022.105183

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: KI67 (1:200, Abcam ab16667); CYCLIN D1 (1:100 for staining, 1:2000 for Western blot, Abcam ab16663); c-MYC (1:1000, MilliporeSigma C3956); V5 (1:5000 for Western blot, 1:250 for immunoprecipitation, Invitrogen R960-25); HA (1:3,000, Sigma H9658); WNT5A (1:2000 for Western blot, 1:100 for immunoprecipitation, Proteintech 55184-1-AP); WNT5B (1:1000, Abclonal A8313); WNT1 (1:1000, Proteintech 27935-1-AP); E-CADHERIN (1:1000, BD 610181); α-TUBULIN (1:2,000, Santa Cruz sc-5286); HSP90 (1:1,000, Abcam ab13492); CALNEXIN (1:5000, Enzo ADI-SPA-860-F); SEL1L (1:2,000, Abcam ab78298); HRD1 (1:300, kindly gifted by Dr. Richard Wojcikiewicz, SUNY Upstate Medical University for Western blot; 1:1000, Sigma HPA024300 for immunostaining of human liver tumors).

Techniques: Western Blot, Plasmid Preparation, Microarray, Recombinant, Purification, Mutagenesis, Software, Expressing

Journal: iScience

Article Title: SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer

doi: 10.1016/j.isci.2022.105183

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: KI67 (1:200, Abcam ab16667); CYCLIN D1 (1:100 for staining, 1:2000 for Western blot, Abcam ab16663); c-MYC (1:1000, MilliporeSigma C3956); V5 (1:5000 for Western blot, 1:250 for immunoprecipitation, Invitrogen R960-25); HA (1:3,000, Sigma H9658); WNT5A (1:2000 for Western blot, 1:100 for immunoprecipitation, Proteintech 55184-1-AP); WNT5B (1:1000, Abclonal A8313); WNT1 (1:1000, Proteintech 27935-1-AP); E-CADHERIN (1:1000, BD 610181); α-TUBULIN (1:2,000, Santa Cruz sc-5286); HSP90 (1:1,000, Abcam ab13492); CALNEXIN (1:5000, Enzo ADI-SPA-860-F); SEL1L (1:2,000, Abcam ab78298); HRD1 (1:300, kindly gifted by Dr. Richard Wojcikiewicz, SUNY Upstate Medical University for Western blot; 1:1000, Sigma HPA024300 for immunostaining of human liver tumors).

Techniques: Western Blot, Virus, Plasmid Preparation, Microarray, Recombinant, Purification, Mutagenesis, Software, Gene Expression

Journal: Nature Communications

Article Title: Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis

doi: 10.1038/s41467-020-17027-5

Figure Lengend Snippet: a , b Complete FES S700C inhibition at 100 nM WEL028 in HL-60 neutrophils. Cells were differentiated into neutrophils (1 µM ATRA, 1.25% DMSO, 72–96 h), treated with WEL028 (100 nM, 1 h) and lysates were post-labeled (1 µM WEL033). Band intensities normalized to vehicle-treated control ( n = 3). c Identification of WEL028 (100 nM) kinase targets in FES S700C HL-60 neutrophils using chemical proteomics. Kinases with >2-fold enrichment compared to vehicle control ( q < 0.05) were designated as targets (means of fold enrichment, n = 4). d – e WEL028 reduces phagocytic uptake in FES S700C but not WT neutrophils. Treatment as in ( a )–( b) , followed by addition of GFP-expressing E. coli B834 (MOI = 30, 1 h, 37 °C) and flow cytometry analysis ( n = 5). Phagocytic index: fraction GFP-positive cells (number of phagocytic cells) multiplied by GFP MFI (number of phagocytized bacteria). f SYK Y352 as proposed FES phosphorylation site (bold red), based on phosphorylation of homologous ZAP70 peptide (PamChip ® microarray, n = 3). g FES phosphorylates SYK Y352 in situ. U2OS cells co-expressing FES S700C -FLAG and SYK-V5 were incubated with vehicle or WEL028 (200 nM, 1 h). Lysates were labeled (250 nM WEL033) and analyzed by in-gel fluorescence and immunoblot ( n = 3). h SYK interacts with FES. Co-transfected U2OS cells were incubated as in ( g ), followed by anti-FLAG immunoprecipitation and immunoblot analysis ( n = 3). i Phagocytosis of E. coli by HL-60 neutrophils depends on FES, SYK and PLCγ2 activity and actin polymerization. Incubations as in ( d )–( e ) (WEL028: 100 nM, R406: 1 µM, U-73122: 5 µM, cytochalasin D: 10 µM; n = 5). j Phosphorylation of SYK Y352 and downstream substrates is inhibited by WEL028 in infected FES S700C but not WT neutrophils, and absent in FES KO neutrophils. Inhibitor incubations as in ( d )-( e ), followed by addition of GFP-expressing E. coli B834 (MOI = 30, 2 min, 37 °C) and immunoblot analysis ( n = 3). Data represent means ± SEM. Statistical analysis: ANOVA with Holm-Sidak’s multiple comparisons correction: *** P < 0.001; * P < 0.05; NS if P > 0.05. Source data are provided as Source Data file.

Article Snippet: Primary antibodies: monoclonal mouse anti-FLAG M2 (1:5000, Sigma Aldrich, F3156), monoclonal anti-V5 (1:5000, Thermo Fisher, R960–25), monoclonal mouse anti-β-actin (1:1000, Abcam, ab8227), polyclonal rabbit anti-Lamin B1 (1:5000, Thermo Fisher, PA5-19468), polyclonal rabbit anti-phospho-FES Y713 (1:1000, Thermo Fisher, PA5-64504), monoclonal rabbit anti-FES (1:1000, Cell Signaling Technology (CST), #85704), polyclonal rabbit anti-phospho-SYK Y352 (1:1000, CST, #2701), monoclonal rabbit anti-SYK (1:1000, CST, #13198), polyclonal rabbit anti-phospho-HS1 Y397 (1:1000, CST, #4507), polyclonal rabbit anti-HS1 (1:1000, CST, #4503), polyclonal rabbit anti-phospho-PLCγ2 Y1217 (1:1000, CST, #3871), polyclonal rabbit anti-PLCγ2 (1:1000, CST, #3872).

Techniques: Inhibition, Labeling, Control, Expressing, Flow Cytometry, Bacteria, Phospho-proteomics, Microarray, In Situ, Incubation, Fluorescence, Western Blot, Transfection, Immunoprecipitation, Activity Assay, Infection, IF-P

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: Lack of HIF-P4H-1 affects the expression of several genes involved in inflammatory and apoptotic pathways. ( a ) Heatmap analysis of microarray data shows that expression of several genes involved in proinflammatory pathways is downregulated in Hif-p4h-1 −/− MEFs relative to wt after 24 h culture in normoxia (21% O 2 ) and hypoxia (1% O 2 ). A comparison between one set of samples is shown in the heatmap. The heatmap was generated by Chipster using Pearson correlation as a distance measure and average linkage for constructing the dendrogram. The red color represents upregulated genes and the green color represents downregulated genes. The values in the color key represent the expression levels between the samples. ( b ) qPCR analysis verifies that the expression of genes for the low affinity III receptor of the Fc fragment of IgG ( Fcgr3 ), the α and γ chains of the complement C1q subcomponent ( C1qa , C1qc ) and the C-X-C motif chemokine 10 ( Cxcl10 ) is downregulated in Hif-p4h-1 −/− MEFs relative to wt in normoxia and hypoxia. ( c ) qPCR analysis shows that expression of the pro-apoptotic genes for the p53 apoptosis effector related to peripheral myelin protein 22 ( Perp ) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 ( Bnip3 ) is upregulated in Hif-p4h-1 −/− MEFs relative to wt. ( b , c ) data are presented as mean ± s.d., n = 3–4 individual Hif-p4h-1 −/− and wt MEF isolates analysed in triplicates. * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test.

Article Snippet: The primary antibodies and dilutions used in Western blotting were anti-RelA (MAB3026 1:1000, Millipore), anti-p-Rel-A (Ser 536, 3033 1:500, Cell Signaling), anti-histone H3 (ab18521 1:1000, Abcam), anti-NF-κB p105/p50 (E381 1:1000, Millipore), anti-caspase-3 (9622 1:500, Cell Signaling), anti-cleaved caspase-3 (Asp175 1:200, Cell Signaling), anti-p53 (CM5 1:500, Vector Laboratories, or PAb421 1:2000, Calbiochem, or DO-1 1:2000, Santa Cruz Biotechnology), anti-MDM2 (NBP2-17247 1:1000, Novus Biologicals), anti-HIF1α (NB100-105 1:500, Novus Biologicals), anti-HIF-P4H-1 (BL525 1:500, Bethyl Laboratories, or TA337938 1:300, Origene), anti-V5 (1:500, Life Technologies), anti-Flag M2 (1:1000, Sigma), anti-HA (SAB1305536 1:500 Sigma), anti-hydroxyproline (ab37067 1:200, Abcam), anti-phospho-p53 Ser15 (9284 1:500, Cell Signaling), anti-β-actin (NB600-501 1:20 000, Novus Biologicals) and anti-α-tubulin (T6199 1:20 0000, Sigma), and the secondary antibodies goat anti-rabbit horse radish peroxidase (HRP)-conjugate (1:20 000 Dako) and goat anti-mouse HRP (1:20 000, Dako).

Techniques: Expressing, Microarray, Comparison, Generated, Two Tailed Test

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: Lack of HIF-P4H-1 increases cell death. ( a – c ) Analysis of relative caspase 3/7 activity ( a ), caspase-3 and cleaved caspase-3 ( b ), and p53 ( c ) protein amounts in wt and Hif-p4h-1 −/− MEFs treated with or without 100 ng/ml LPS for 24 h. ( d ) Analysis of viability of wt and Hif-p4h-1 −/− MEFs treated with or without 5 mg/ml of LPS for 48h. Quantitation of cell viability based on 7-actinomycin D staining and FACS analysis is shown. ( e ) Analysis of relative caspase 3/7 activity in Hif-p4h-1 −/− MEFs transfected with empty vector (EV) or a vector encoding V5-tagged human recombinant HIF-P4H-1 (OE). ( f ) Analysis of viability of wt and Hif-p4h-1 −/− MEFs treated with or without 2 μM staurosporine for 24 h. ( g ) Analysis of viability of Hif-p4h-1 −/− MEFs transfected with empty vector (EV) or a vector overexpressing (OE) V5-tagged human recombinant HIF-P4H-1 and treated with 2 μM staurosporine for 24 h. Quantitation of cell viability based on 7-actinomycin D staining and FACS analysis is shown. ( h ) Western blot analysis of p53, caspase-3, cleaved caspase-3 and HIF-P4H-1 in wt and Hif-p4h-1 −/− MEFs treated with or without 10 μg/ml cisplatin for 6 h. ( i ) Western blot analysis of caspase-3, cleaved caspase-3 and HIF-P4H-1 in wt MEFs treated with or without 10 μg/ml cisplatin and 50 µM FG4497 for 6 h. ( j ) Analysis of viability of Hif-p4h-1 −/− and wt MEFs treated with or without 50 μg/ml cisplatin and 100 μM PFT-α for 24 h. Quantitation of cell viability based on dynamic imaging of live and dead cells in the IncuCyte TM zoom live-cell imaging system is shown. ( k ) Western blot analysis of HIF-P4H-1 in wt HCT-116 cells treated with or without 100 ng/ml LPS and 50 µM FG4497 for 6 h. Data are presented as representative Western blots and as mean ± s.d., n = 3 individual MEF isolates or experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: The primary antibodies and dilutions used in Western blotting were anti-RelA (MAB3026 1:1000, Millipore), anti-p-Rel-A (Ser 536, 3033 1:500, Cell Signaling), anti-histone H3 (ab18521 1:1000, Abcam), anti-NF-κB p105/p50 (E381 1:1000, Millipore), anti-caspase-3 (9622 1:500, Cell Signaling), anti-cleaved caspase-3 (Asp175 1:200, Cell Signaling), anti-p53 (CM5 1:500, Vector Laboratories, or PAb421 1:2000, Calbiochem, or DO-1 1:2000, Santa Cruz Biotechnology), anti-MDM2 (NBP2-17247 1:1000, Novus Biologicals), anti-HIF1α (NB100-105 1:500, Novus Biologicals), anti-HIF-P4H-1 (BL525 1:500, Bethyl Laboratories, or TA337938 1:300, Origene), anti-V5 (1:500, Life Technologies), anti-Flag M2 (1:1000, Sigma), anti-HA (SAB1305536 1:500 Sigma), anti-hydroxyproline (ab37067 1:200, Abcam), anti-phospho-p53 Ser15 (9284 1:500, Cell Signaling), anti-β-actin (NB600-501 1:20 000, Novus Biologicals) and anti-α-tubulin (T6199 1:20 0000, Sigma), and the secondary antibodies goat anti-rabbit horse radish peroxidase (HRP)-conjugate (1:20 000 Dako) and goat anti-mouse HRP (1:20 000, Dako).

Techniques: Activity Assay, Quantitation Assay, Staining, Transfection, Plasmid Preparation, Recombinant, Western Blot, Imaging, Live Cell Imaging, Two Tailed Test

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: Lack of HIF-P4H-1 increases the amount of p53. ( a ) Western blot analysis of p53 in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h. ( b , c ) Western blot analysis of p53 in Hif-p4h-1 and scrambled (Scrm) siRNA transfected HEK293 cells ( b ) and wt MEFs treated with 50 µM FG4497 for 24 h ( c ). ( d ) qPCR analysis of p53 mRNA in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h. ( e) Analysis of p53 protein turnover rate. Hif-p4h-1 −/− and wt MEFs were treated with 200 μg/ml of cycloheximide for the indicated time points and whole cell lysates were blotted for p53. ( f ) Western blot analysis of MDM2 in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h. ( g ) Western blot analysis of p53 and MDM2 in wt MEFs treated with 10 μM nutlin-3a for 24 h with or without overexpression of human HIF-P4H-1 (OE). ( h , i ) Western blot analysis of HIF1α in wt and Hif-p4h-1 −/− MEFs cultured in 21% or 1% O 2 for 24 h ( h ) and in scrambled and Hif-p4h-1 siRNA transfected HEK293 cells ( i ). ( j ) Western blot analysis of ubiquitination of p53 in Hif-p4h-1 and scrambled siRNA transfected HEK293 cells. The cells were transfected with HA-ubiquitin and endogenous p53 was immunoprecipitated followed by Western blotting with anti-HA and anti-P53 antibodies. ( k ) Western blot analysis of p53 in Hif-p4h-1 −/− and wt MEFs were treated with or without 10 μM MG132 for 5 h. Data are presented as representative Western blots and as mean ± s.d., n = at least 3 individual MEF isolates or experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: The primary antibodies and dilutions used in Western blotting were anti-RelA (MAB3026 1:1000, Millipore), anti-p-Rel-A (Ser 536, 3033 1:500, Cell Signaling), anti-histone H3 (ab18521 1:1000, Abcam), anti-NF-κB p105/p50 (E381 1:1000, Millipore), anti-caspase-3 (9622 1:500, Cell Signaling), anti-cleaved caspase-3 (Asp175 1:200, Cell Signaling), anti-p53 (CM5 1:500, Vector Laboratories, or PAb421 1:2000, Calbiochem, or DO-1 1:2000, Santa Cruz Biotechnology), anti-MDM2 (NBP2-17247 1:1000, Novus Biologicals), anti-HIF1α (NB100-105 1:500, Novus Biologicals), anti-HIF-P4H-1 (BL525 1:500, Bethyl Laboratories, or TA337938 1:300, Origene), anti-V5 (1:500, Life Technologies), anti-Flag M2 (1:1000, Sigma), anti-HA (SAB1305536 1:500 Sigma), anti-hydroxyproline (ab37067 1:200, Abcam), anti-phospho-p53 Ser15 (9284 1:500, Cell Signaling), anti-β-actin (NB600-501 1:20 000, Novus Biologicals) and anti-α-tubulin (T6199 1:20 0000, Sigma), and the secondary antibodies goat anti-rabbit horse radish peroxidase (HRP)-conjugate (1:20 000 Dako) and goat anti-mouse HRP (1:20 000, Dako).

Techniques: Western Blot, Cell Culture, Transfection, Over Expression, Ubiquitin Proteomics, Immunoprecipitation, Two Tailed Test

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: HIF-P4H-1 interacts with p53. ( a ) Immunoprecipitation of endogenous p53 from wt MEFs treated with 10 μM MG132 for 4 h before cell lysis. Co-immunoprecipitation of endogenous HIF-P4H-1 was analyzed by Western blotting with an anti-HIF-P4H-1 antibody. ( b ) HEK293 cells were transfected with a plasmid encoding full-length p53 together with a plasmid encoding V5-tagged human HIF-P4H-1 (OE) or an empty vector (EV) and analysed by Western blotting with anti-p53 and anti-V5 antibodies. ( c ) HEK293 cells were transfected as in ( b ) treated with 10 μM MG132 for 4 h and immunoprecipitated with an anti-V5 antibody. Co-immunoprecipitation of p53 was analysed by Western blotting with an anti-p53 antibody. ( d ) HEK293 cells were transfected with a plasmid encoding a deleted form of p53 that lacks amino acid residues 75–207 (p53Δ) together with a plasmid encoding V5-tagged human HIF-P4H-1 (OE) or an empty vector and analysed by Western blotting with anti-p53 and anti-V5 antibodies. ( e ) HEK293 cells were transfected as in d , treated with 10 μM MG132 for 4 h and immunoprecipitated with an anti-V5 antibody. Co-immunoprecipitation of p53 was analysed by Western blotting with an anti-p53 antibody. Data are presented as representative Western blots and as mean ± s.d., n = at least 3 individual experiments. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: The primary antibodies and dilutions used in Western blotting were anti-RelA (MAB3026 1:1000, Millipore), anti-p-Rel-A (Ser 536, 3033 1:500, Cell Signaling), anti-histone H3 (ab18521 1:1000, Abcam), anti-NF-κB p105/p50 (E381 1:1000, Millipore), anti-caspase-3 (9622 1:500, Cell Signaling), anti-cleaved caspase-3 (Asp175 1:200, Cell Signaling), anti-p53 (CM5 1:500, Vector Laboratories, or PAb421 1:2000, Calbiochem, or DO-1 1:2000, Santa Cruz Biotechnology), anti-MDM2 (NBP2-17247 1:1000, Novus Biologicals), anti-HIF1α (NB100-105 1:500, Novus Biologicals), anti-HIF-P4H-1 (BL525 1:500, Bethyl Laboratories, or TA337938 1:300, Origene), anti-V5 (1:500, Life Technologies), anti-Flag M2 (1:1000, Sigma), anti-HA (SAB1305536 1:500 Sigma), anti-hydroxyproline (ab37067 1:200, Abcam), anti-phospho-p53 Ser15 (9284 1:500, Cell Signaling), anti-β-actin (NB600-501 1:20 000, Novus Biologicals) and anti-α-tubulin (T6199 1:20 0000, Sigma), and the secondary antibodies goat anti-rabbit horse radish peroxidase (HRP)-conjugate (1:20 000 Dako) and goat anti-mouse HRP (1:20 000, Dako).

Techniques: Immunoprecipitation, Lysis, Western Blot, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways

doi: 10.1038/s41598-017-17376-0

Figure Lengend Snippet: p53 is hydroxylated at Pro142. ( a ) Flag-tagged p53 was immunoprecipitated from HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h, separated by SDS-PAGE, trypsinized and analyzed by LC-MS. Mass signals extracted from the high energy trace of the MS e measurement assigned to the peptide TCPVQLWVDSTPPPGTR (140–156) are shown. Low molecular weight area for the oxidized (i) and unmodified (ii) peptide with the b4 ions was used to assign hydroxylation to Pro142. Detailed information on all found fragment ions are provided in Supplementary tables and . ( b ) [2,3,4,5- 3 H] proline-labeled Flag-tagged p53 was produced in wt HCT-116 cells treated with 10 μg/ml cisplatin and 10 μM MG132 with or without 50 μM FG4497 for 6 h. Flag-p53 was immunoprecipitated and the amount of 4-hydroxy[ 3 H]proline formed was measured by a radiochemical method. The data are given as the amount of 4-hydroxyproline residues/50 000 proline residues. ( c ) HCT-116 cells were transfected with scrambled or HIF-P4H-1 siRNA and treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. Endogenous p53 was immunoprecipitated and analysed by Western blotting with antibodies agains hydroxyproline and p53. ( d ) Wt and HIF1A −/− HCT-116 cells were treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h with or without 50 μM FG4497. Endogenous p53 was immunoprecipitated and analysed by Western blotting with antibodies against hydroxyproline, P(Ser15)-p53 and p53. ( e , f ) Flag-tagged wt, Pro12Ala/Pro13Ala and Pro142Ala p53 were overexpressed (OE) in HCT-116 cells with or without V5-tagged HIF-P4H-1 ( e ) and with or without 50 μM FG4497 for 6 h ( f ) and the amount of p53 and HIF-P4H-1 was analysed by Western blotting with anti-Flag and anti-V5 antibodies, respectively. ( g ) Flag-tagged wt and Pro142Ala p53 and V5-tagged HIF-P4H-1 were overexpressed in HEK293 cells and treated with 10 μM MG132 for 6 h. Flag-tagged p53 proteins were immunoprecipitated and co-immunoprecipitation of HIF-P4H-1 was analysed by Western blotting with an anti-V5 antibody. ( h ) Flag-tagged wt and Pro142Ala p53 were overexpressed in HCT-116 cells and treated with 10 μg/ml cisplatin and 10 μM MG132 for 6 h. Flag-p53 was immunoprecipitated and hydroxylation was analysed by Western blotting with an anti-hydroxyproline antibody. ( i ) V5-tagged HIF-P4H-1 was overexpressed (OE) in wt and HIF1A −/− HCT-116 cells treated with 10 μM MG132 for 6 h. Endogenous p53 was immunoprecipitated and co-immunoprecipitation of HIF-P4H-1 was analysed by Western blotting with an anti-V5 antibody. Data are presented as representative Western blots and as mean ± s.d., n = at least 3 individual experiments in ( b – i ). * P < 0.05, ** P < 0.01 and *** P < 0.001, two-tailed Student’s t -test. Unprocessed original scans of blots are shown in Supplementary Fig. .

Article Snippet: The primary antibodies and dilutions used in Western blotting were anti-RelA (MAB3026 1:1000, Millipore), anti-p-Rel-A (Ser 536, 3033 1:500, Cell Signaling), anti-histone H3 (ab18521 1:1000, Abcam), anti-NF-κB p105/p50 (E381 1:1000, Millipore), anti-caspase-3 (9622 1:500, Cell Signaling), anti-cleaved caspase-3 (Asp175 1:200, Cell Signaling), anti-p53 (CM5 1:500, Vector Laboratories, or PAb421 1:2000, Calbiochem, or DO-1 1:2000, Santa Cruz Biotechnology), anti-MDM2 (NBP2-17247 1:1000, Novus Biologicals), anti-HIF1α (NB100-105 1:500, Novus Biologicals), anti-HIF-P4H-1 (BL525 1:500, Bethyl Laboratories, or TA337938 1:300, Origene), anti-V5 (1:500, Life Technologies), anti-Flag M2 (1:1000, Sigma), anti-HA (SAB1305536 1:500 Sigma), anti-hydroxyproline (ab37067 1:200, Abcam), anti-phospho-p53 Ser15 (9284 1:500, Cell Signaling), anti-β-actin (NB600-501 1:20 000, Novus Biologicals) and anti-α-tubulin (T6199 1:20 0000, Sigma), and the secondary antibodies goat anti-rabbit horse radish peroxidase (HRP)-conjugate (1:20 000 Dako) and goat anti-mouse HRP (1:20 000, Dako).

Techniques: Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Molecular Weight, Labeling, Produced, Transfection, Western Blot, Two Tailed Test