Journal: Cell Genomics

Article Title: Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

doi: 10.1016/j.xgen.2022.100207

Figure Lengend Snippet: A nonsynonymous variant in ERAP1 regulates IAV burden in cells (A) Stratified QQ plot for association with mean IAV reads restricted to RASQUAL 2-step FDR eQTLs (FDR < 0.05; MAF > 0.1). Plotting eQTLs reveals an SNP associated with expression of TNFSF12 that has a lower p value than expected by chance. (B) Stratified QQ plot for association with mean IAV reads restricted to nonsynonymous SNPs. Plotting nonsynonymous variants (MAF >0.1) reveals an SNP in ERAP1 that has a p value lower than expected by chance. (C) Genotype median plot for mean IAV reads as a function of rs27895 genotype. P value from EMMAX and corrected for genomic inflation factor. (D) RNAi against TNFSF12 in either NA19399 (TT for rs12103519) or NA19328 (CC for rs12103519) demonstrates reducing TNFSF12 expression (mean 87% knockdown ±4% [SEM] for NA19328 and mean 73% knockdown ±6% [SEM] for NA19399) does not significantly affect the percentage of IAV-infected cells at 24 h in either LCL. Replicates from 3 separate experiments were normalized to correct for between-experiment variation. p values are from unpaired t tests. (E) RNAi against ERAP1 in either NA19020 (TT for rs27895) or NA19399 (CC for rs27895) demonstrate that reducing ERAP1 expression (mean 80% knockdown ±14% [SEM] for NA19020 and mean 70% knockdown ±8% [SEM] for NA19399) decreases the percentage of IAV-infected cells at 24 h in both LCLs. Replicates from 4 separate experiments were normalized to correct for between-experiment variation. One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s. Replicates from 3 separate experiments were normalized to correct for between-experiment variation. p values are from ordinary one-way ANOVA with Dunnett’s multiple comparisons test using DMSO (vehicle) control. (G) Interaction between ERAP1 residue 346 and an ERAP1 peptide inhibitor. Ribbon diagram of the crystal structure of ERAP1 (light gray, cartoon) bound to a 10-mer peptide inhibitor (purple) (PDB: 6RQX ). The carbon atoms of ERAP1 residue 346 and the sixth position of the peptide are shown as orange-, yellow- and purple-colored spheres, respectively. (H) Wild-type ERAP1 G346 and F6 of the 10-mer peptide inhibitor have multiple favorable side chain-backbone van der Waals contacts. (I) However, steric clash between the ERAP1 side chain and the 10-mer peptide inhibitor occurs when G346 is mutated to an aspartate (carbon atoms colored green) using in silico mutagenesis. (J) Rare rotamers of ERAP1 G346D do not clash with a peptide in which F6 is replace by a glycine but provide no favorable contacts. (K) Even the addition of only a β carbon at position 6 of the 10-mer peptide inhibitor, i.e., an alanine residue, clashes with ERAP1 (G346D). (L) Overexpression of alternative alleles of ERAP1 in A549 cells demonstrates that the T allele of rs27895 (aspartate) increases the percentage of IAV-infected cells at 24 h. 8 biological replicates from 3 separate experiments were normalized to correct for between-experiment variation. p values are from ordinary one-way ANOVA with Tukey’s multiple comparisons test. Overexpression of alternative alleles of ERAP1 was confirmed by western blot. (M) Model of how rs27895 affects ERAP1 ’s transcript and protein and the resulting effect on viral burden. The reference allele of rs27895 encodes cytosine on the genome reference strand and guanine on the transcribed strand. This reference allele encodes glycine at position 346 of ERAP1 and is associated with reduced viral burden in cells. The alternate allele of rs27895 encodes thymine on the genome reference strand and adenine on the transcribed strand. This alternate allele encodes aspartate at position 346 of ERAP1 and is associated with increased viral burden in cells

Article Snippet: One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s.

Techniques: Variant Assay, Expressing, Knockdown, Infection, Control, Residue, In Silico, Mutagenesis, Over Expression, Western Blot

Journal: Cell Genomics

Article Title: Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

doi: 10.1016/j.xgen.2022.100207

Figure Lengend Snippet: A nonsynonymous variant in ERAP1 regulates IAV burden and symptomology in human challenge (A) Flow chart of Prometheus study. (B) rs27895 T allele is associated with IAV burden at day 4 of the Prometheus study. Viral burden was measured by nasal lavage by qPCR using pan IAV M gene primers (see ). p values from EMMAX at each time point for rs27895 are listed below the plot. Mean and SD are plotted with lines connecting means. (C) rs27895 T allele is associated with greater symptomology at days 3–7. Jackson symptomology score was assessed daily. p values from EMMAX at each time point for rs27895 are listed below the plot. Mean and SD are plotted with lines connecting means

Article Snippet: One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s.

Techniques: Variant Assay

Journal: Cell Genomics

Article Title: Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus

doi: 10.1016/j.xgen.2022.100207

Figure Lengend Snippet:

Article Snippet: One experiment involving NA19020 was excluded because no knockdown was detected. p values are from unpaired t tests. (F) Specific ERAP1 inhibitor, ERAP1-IN-1 (CAS: 865273-97-8, MedChemExpress) demonstrates dose-dependent reduction in the percentage of IAV-infected cells at 24 h in A549s.

Techniques: Virus, Recombinant, Over Expression, TaqMan Assay, Software

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: Erap1 (a, c, e) and Tap1 (b, d, e) expression in normal neural stem cells (NSCs, n=3), MP tumors (n=3), MG tumors (n=3), MG tumors overexpressing DNp53 (MG+P) (n=3) and MP tumors overexpressing GFI1 (MP+G) (n=3) were measured by qRT-PCR (a-d) and by western blotting (e); quantification of 3 independent experiments is shown below the western blot. Error bars represent means ± SD. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. p-values were determined by two-sided unpaired t-test. In (a), NS: p=0.21 (MG vs NSC), ** p=0.0019 (MG vs MP). In (b) NS, p=0.33 (MG vs NSC); *** p=1.9E-05. In (c), *** p=1E-04 (MP vs MG) and p=8E-05 (MG vs MG+P). In (d), *** p=2.3E-05 (MP vs MG), ** p=0.003 (MG vs MG+P, Tap1). In (e), * refers to p=0.002 (MP vs MG, Erap1), p=0.0052 (MG vs MG+P,Erap1), p=0.0097 (MP vs MG, Tap1), p=0.0049 (MG vs MG+P, Tap1). (f-g) Analysis of ERAP1 (f) and TAP1 (g) mRNA levels in human Sonic Hedgehog-associated MB with mutant (red) or wild-type (WT, blue)) TP53. Fragments Per Kilobase per Million mapped reads (FPKM) of ERAP1 and TAP1 are shown. p-values were determined by two-sided Wilcoxon rank sum test. Boxplot center lines show median, box limits indicate the 25th and 75th percentiles, lower and upper whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles, respectively. (h) Putative p53 binding sites in the mouse Erap1 and Tap1 promoters. (i-j) Chromatin Immunoprecipitation (ChIP) and PCR analysis of p53 binding site-containing regions in the Erap1 (i) and Tap1 (j) promoters. qPCR results for anti-p53 ChIP (IP p53) or isotype control ChIP (IP Ctl) in MG (n=3) and MP (n=3) tumor cells. p-values were determined by two-sided unpaired t-test * p=0.0046 (Erap1) and ** p=0.0011 (Tap1). (k) Putative p53 binding sites in the human ERAP1 and TAP1 promoters. (l-m) ChIP and PCR analysis of the p53 binding site-containing regions in the ERAP1 (l) and TAP1 (m) promoters. qPCR results for anti-p53 ChIP (IP p53) or isotype control ChIP (IP Ctl) in p53 mutant PDXs (RCMB18, Icb984, BT084) and p53 wild-type PDXs (Icb1299, RCMB40) (n=3 for each tumor type). p-values were determined by two-sided unpaired t-test. * p=0.026 (Icb1299, ERAP1), p=0.01 (Icb1299, TAP1), p=0.002 (RCMB40, TAP1) ; ** p=0.001 (RCMB40, ERAP1).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Control

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: MG (a, c, e, g) and MP (b, d, f, h) tumor cells were treated in vitro for 48h with IFNα (20 ng/mL), IFNγ (20 ng/mL), TNF (50 pg/mL) or LtβRag (1.6μg/mL). MHC-I expression in untreated cells (Ctl, black histograms) and cells treated with IFNα (red, a, b), IFNγ (red, c, d), TNF (green, e, f) or LTβRag (blue, g, h) were analyzed by FACS. Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below each histogram; data points represent MFIs for individual tumor samples. p-values were determined by two-sided unpaired t-test. p-values are indicated on the corresponding graphs. Erap1 (i) and Tap1 (j) mRNA expression in untreated MP tumor cells (Ctl) or MP tumor cells treated with TNF, LTβRag or IFNγ were determined by qRT-PCR (n=3). Data points represent expression values for individual tumor samples; error bars represent the mean ± SD. p-values were determined by two-sided unpaired t-test. In (i), NS, p=0.351;* p=0.006, ***p=0.0004. In (j), NS, p=0.453, * p=0.0086, ** p=0.003. (k) Tap1 and Erap1 protein levels were assayed by western blotting. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. Relative protein levels of Erap1 (l) and Tap1 (m) normalized to actin are shown for three independent samples. Error bars represent mean ± SD; data points represent expression values for individual tumor samples. p-values were determined by two-sided unpaired t-test. In (l), NS p=0.133, * p=0.0025, ** p=0.0004 ; In (m), NS = 0.378, * p=0.029, ** p=0.0116.

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: In Vitro, Expressing, Fluorescence, Quantitative RT-PCR, Western Blot

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: (a-d) MG tumor cells were transduced with control shRNA (shCtl) or shRNAs targeting Erap1 (shErap1#1, shErap1#2). Knockdown efficiency was determined by western blotting in three independent tumor samples (a). MHC-I expression was determined by FACS in control cells (shCtl, black) and Erap1 knockdown cells (shErap1, red). Western blots are cropped at the molecular weights for Erap1 (120 kDa) and β-actin (42 kDa); original blots are available in Source Data. (b). Quantification of the mean fluorescence intensity (MFI) values for three independent tumors is shown below each histogram; data points represent MFIs for individual tumor samples. p-values, determined by two-sided unpaired t-test, are indicated on each bar graph. (c-d) Erap1 knockdown cells were transplanted into aB6 mice. Bioluminescence imaging of representative mice (c) and survival curves (n=6) (d) are shown. p-values for the difference in survival between shErap1 and shCtl were determined using the two-sided log-rank (Mantel-Cox) test. * p=0.024; ** p= 0.0075. (e-i) MP tumor cells were transduced with empty vector (vect) or vectors encoding Erap1, Tap1 or both. Erap1 (e) and Tap1 (f) expression levels were assessed by western blotting in three independent tumor samples. Western blots are cropped at the molecular weights for Erap1 (120 kDa), Tap1 (68kDa) and β-actin (42 kDa); original blots are available in Source Data. (g) Analysis of MHC-I expression by FACS in control cells (vect, black) and Erap1 + Tap1 overexpressing cells (pink). Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below the histogram; data points represent MFIs for individual tumor samples. The p-value was determined by two-sided unpaired t-test. (h, i) MP tumor cells transduced with control vector (vect, black), Tap1 (green), Erap1 (blue) or Erap1 + Tap1 (pink) were orthotopically transplanted into aB6 mice. Bioluminescence imaging of representative mice (h) and survival curves (n=6 per group) (i) are shown. p-values were determined by the two-sided log-rank (Mantel-Cox) test. NS (Ctl vs. Tap1), p=0.79; * (Ctl vs. Erap1) p=0.0047; ** (Ctl vs. Erap1+Tap1) p=0.0005. Mice carrying MP tumors expressing Erap1 + Tap1 survive significantly longer than mice carrying tumors expressing Erap1 alone (p=0.0016 for Erap1 vs. Erap1 + Tap1) or Tap1 alone (p = 0.0005 for Tap1 vs. Erap1 + Tap1).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Transduction, Control, shRNA, Knockdown, Western Blot, Expressing, Fluorescence, Imaging, Plasmid Preparation

Journal: Nature neuroscience

Article Title: Tumor Necrosis Factor Overcomes Immune Evasion in p53-Mutant Medulloblastoma

doi: 10.1038/s41593-020-0628-4

Figure Lengend Snippet: (a, b) FACS analysis of MHC-I expression in MP tumor cells that were untreated (Ctl, black histograms) or treated for 48h with TNFR1 agonist (0.5μg/mL, purple histogram, a) or TNFR2 agonist (1.67nM, red histogram, b). (c) MP tumor cells generated from TNFR2 knockout (TNFR2 KO) mice were treated for 48h with no stimulus (Ctl, black histogram) or with TNF (50 pg/mL, green histogram) and then analyzed by FACS. Quantification of the mean fluorescence intensity (MFI) for three independent experiments is shown below each histogram; data points represent MFIs for individual tumor samples. p-values were determined by two-sided unpaired t-test. p-values are indicated on the corresponding graphs. (d-f) MP tumor cells were untreated (NT) or treated with TNF for 15’, 30’ or 1h. Expression of RelA and RelB protein were assessed by western blotting in nuclear extracts (d) and in total cellular protein extracts (e). Histone H3 and GAPDH were used as controls for nuclear and total extracts respectively. Western blots are cropped at the molecular weights for RelA (65 kDa), RelB (68kDa), Histone H3 (17kDa) and GAPDH (37 kDa); original blots are available in Source Data. (f) Quantification of Western blots for RelA (black) and RelB (blue) protein levels in nucleus relative to levels in total extract at 0’, 15’, 30’ and 60’ for three independent experiments. p-values were determined by two-sided unpaired t-test. For RelA, * p=0.049 (15’) ; ***p=0.0032 (30’) ; **p=0.0043 (1h); for RelB: NS, p=0.088 (15’) ; *p=0.037 (30’) ; NS p=0.073 (1h). (g, i) Putative binding sites for the RelA-p50 heterodimer in the mouse Erap1 and Tap1 promoters. (h, j) MP tumor cells were treated for 1h with no stimulus (ctl), TNF or LtβRag. Chromatin Immunoprecipitation (ChIP) was performed using antibodies specific for RelA (h) or p50 (j), and precipitates were analyzed by qPCR for the presence of the RelA-p50 binding site-containing regions of the Erap1 and Tap1 promoters. Data represent the mean fold-induction ± SD. p values were determined by two-sided unpaired t test. In (h), * p=0.0034, **p=0.003, ***p=2.6E-06 (TNF, Erap1), p=1E-04 (TNF, Tap1). (k) MP tumor cells were transduced with control shRNA (shCtl) or shRNAs targeting RelA (shRelA#832, shRelA#833), p50 (shp50#484, shp50#485) or RelB (shRelB#494, shRelB#495). MHC-I expression was determined by FACS in control cells (shCtl) and RelA, p50 or RelB knockdown cells treated in vitro for 48h with vehicle (DMSO, ctl), TNF or LtβRag. Data shown represent quantification of mean fluorescence intensity (MFI) for three independent experiments; data points represent MFIs for individual tumor samples. p values were determined by two-sided unpaired t test. shCtl : ** p=0.0019, *** p=1.1E-05; shRelA (#832): NS, Not significant p=0.063 (TNF) and p=0.098 (LtβRag); shRelA (#833): *p=0.035, **p=0.009; shp50 (#484): NS, Not significant p=0.341 (TNF), p=0.125 (LtβRag); shp50 (#485): NS, p=0.601, *p=0.049 ; shRelB (#494) *** p=1.8E-05 (TNF), p=2.3E-04 (LtβRag); shRelB (#495): *** p=9E-06 (TNF), p=1.6E-05 (LtβRag).

Article Snippet: For GOF experiments, the Erap1 construct (pFB-CT10HF-LIC-ERAP1, Addgene, plasmid #39174), a gift from Nicola Burgess-Brown and the Tap1 construct (MGC-TAP1 from Dharmacon) were used to clone Erap1 and Tap1 respectively into pMSCV-IRES-GFP.

Techniques: Expressing, Generated, Knock-Out, Fluorescence, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Transduction, Control, shRNA, Knockdown, In Vitro

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 positively regulates the Hh pathway at postreceptor level. a Luciferase activity of NIH3T3 Shh-Light II cells treated for 24 h with SAG and increasing amounts of Leu-SH or DTT as control. b , c Quantitative real-time PCR (qRT-PCR) ( b ) and representative immunoblotting ( c ) analyses of Gli1 expression in the NIH3T3 murine fibroblasts transduced with lentiviral vectors encoding either control shRNA (shCTRL) or ERAP1 shRNA (shERAP1#1 and shERAP1#2) and treated with SAG or DMSO for either 24 or 48 h. In c ERAP1 expression was also evaluated and actin was used as loading control. d , f qRT-PCR analysis of Hh target genes expression in Ptch −/− ( d ) and SuFu −/− MEFs ( f ) both treated with Leu-SH (30 μM) or DTT as control. e Representative model of the constitutive activation of Smo or Gli1 in Ptch −/− and SuFu −/− MEFs, respectively. g , h qRT-PCR analysis of Hh target genes expression in Ptch −/− ( g ) and SuFu −/− MEFs ( h ) transduced with shCTRL or shERAP1 constructs. Data in b , d , f , g , and h are normalized to endogenous GAPDH and HPRT controls and expressed as the fold change respect to the control sample value. All data represent the mean of three independent experiments. Mean ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001 calculated with two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transduction, shRNA, Activation Assay, Construct

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 activates Hh signaling by impairing βTrCP protein expression. a – f Representative immunoblotting analyses of the indicated proteins in MEFs transfected with increasing amounts of vector encoding ERAP1 ( a , d ) or treated for 24 h with Leu-SH at the indicated concentration ( b , e ), or SAG (200 nM) and Leu-SH (30 μM) ( c ), or DTT as control. In f MEFs were transduced with shCTRL or shERAP1 and transfected with a vector encoding ERAP1. Actin ( a – c , f ) and tubulin ( d , e ) were used as loading controls. g ERAP1, Gli1 and βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 in the presence of small interfering RNAs (siRNAs) to a non-relevant mRNA (siCTRL) or murine βTrCP mRNA (siβTrCP). h βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 and treated with cycloheximide (CHX, 100 µg/mL) at different time points. Densitometry analysis of actin-normalized βTrCP values of three independent experiments is shown (right panel). i , j Endogenous βTrCP was immunoprecipitated from MEFs expressing the indicated proteins and treated with MG132 (50 μM) for 4 h ( i ) or increasing doses of Leu-SH for 24 h ( j ), followed by immunoblotting with an anti-HA antibody to detect conjugated HA-Ub. Blots were both reprobed with a βTrCP antibody. Bottom ERAP1 and βTrCP protein levels in total cell lysate. Actin was used as loading control. k Immunoblotting (upper panel) and densitometric analysis (lower panel) of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs and treated after 24 h with increasing amount of Leu-SH for 24 h. l Immunoblotting analysis of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs transduced with shCTRL or shERAP1. Actin was used as loading control

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Concentration Assay, Transduction, Immunoprecipitation

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 promotes βTrCP ubiquitylation by interacting with USP47. a – d MEFs were transfected with ERAP1 and/or Flag-USP47. Interaction between USP47 and ERAP1 was detected by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. e MEFs transfected with ERAP1 were stained with anti-ERAP1 and anti-USP47 antibodies. Green and red, USP47 and ERAP1 expressing cells, respectively. Nuclei were counter stained with Hoechst (Blue). Magnification ×60; Bars: 5 μm. Representative images from three independent experiments. f βTrCP and Gli steady state in USP47 +/+ and USP47 −/− MEFs. g βTrCP protein level in USP47 +/+ , USP47 −/− and USP47 −/− Flag-USP47 transfected MEFs. h βTrCP half-life in USP47 +/+ vs. USP47 −/− MEFs treated with CHX (100 µg/mL) at the indicated times. i βTrCP protein levels in MEFs transfected with empty vector as control or Flag-USP47 and treated with CHX (100 µg/mL) at different time points. j Gli1 protein levels in Ptch −/− MEFs transfected with empty vector as control or Flag-USP47 and treated after 24 h with CHX (100 µg/mL) for different time points. In g – j densitometric analysis of βTrCP and Gli1 protein levels of three independent experiments are shown (right panels). k MEFs were transfected with HA-Ub and increasing amount of ERAP1 in the presence or absence of Flag-USP47. Endogenous βTrCP was immunoprecipitated with an anti-βTrCP antibody and the ubiquitylated forms were revealed with an anti-HA antibody (upper panel). The blot was reprobed with an anti-βTrCP antibody. Flag-USP47, ERAP1 and βTrCP total protein levels are shown (lower panel). l MEFs were transfected with Flag-USP47 and increasing amount of ERAP1. Interaction between Flag-USP47 and endogenous βTrCP was assessed by immunoprecipitation and immunoblotting with the indicated antibodies. Actin was used as loading control. m MEFs were transfected with Flag-USP47 and treated for 24 h with Leu-SH at the indicated concentration. Interaction between Flag-USP47 and endogenous βTrCP was detected as described in l . Densitometric analysis of the Flag-USP47/βTrCP binding ratio representative of three independent experiments is shown (right panel). n MEFs were transduced with shCTRL or shERAP1 and transfected with Flag-USP47. Interaction between Flag-USP47 and endogenous βTrCP was assessed as described in l

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Transfection, Immunoprecipitation, Western Blot, Staining, Expressing, Plasmid Preparation, Concentration Assay, Binding Assay, Transduction

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 impairs Hh-dependent growth of cerebellar granule cell progenitors. a H&E and immunohistochemical staining of Ki67, ERAP1 and Gli1 in the outer EGL during mouse cerebellum development. Magnification ×40. Scale bars represent 50 μm. b , c GCPs were isolated from 4-day-old mice and treated with either SAG alone or in combination with increasing doses of Leu-SH for 24 h. BrdU uptake ( b ) and mRNA levels of Gli1 ( c ) are shown. d , i GCPs isolated from 4-day-old mice were infected with lentiviral particles encoding for shERAP1 ( d – f ) or ERAP1 ( g – i ) and the corresponding controls, respectively. The percentage of BrdU uptake ( d , g ), mRNA ( e , h ), and protein levels ( f , i ) of Gli1 are shown. Results in c , e , h were normalized to endogenous GAPDH and HPRT controls and expressed as described in Fig. legend, and represent the mean of three independent experiments. In f and i, actin was used as loading control. Mean ± S.D. * P < 0.05; ** P < 0.01 determined with two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Immunohistochemical staining, Staining, Isolation, Infection

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 impinges Hh-dependent tumor cell growth in vitro. a – f Primary cell cultures from Math1-cre/Ptc C/C mice MBs were treated with different amounts of Leu-SH. a , b Cells were counted with trypan blue at the indicated time points to evaluate the growth rate of viable cells ( a ) and the percentage of cell death ( b ). c Cleaved Caspase-3 protein levels in cells treated with Leu-SH at the indicated concentration for 24 h. d – f Percentage of BrdU uptake ( d ) and Gli1 mRNA ( e ), and protein ( f ) expression in MB cells treated with Leu-SH at the indicated concentrations for 24 h. g MB Stem-Like Cells (MB-SLCs) from Math1-cre/Ptc C/C mice were treated with Leu-SH as in (a) and counted with trypan blue at the indicated time points. h MB-SLCs were dissociated and treated with the indicated concentrations of Leu-SH or DTT as control. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was evaluated. The self-renewal MB-SLCs capability is expressed as percentage of neurosphere-forming cells (right). Representative bright field images of tumor neurospheres after Leu-SH treatment are shown (left). Scale bar 100 µM. i , j mRNA and protein expression levels of Hh target genes of MB-SLCs treated with the indicated concentrations of Leu-SH for 24 h. Actin was used as loading control. Results in e , i were normalized to endogenous GAPDH and HPRT controls and expressed as described in Fig. legend. All data are representative of three independent experiments. Mean ± S.D. * P < 0.05; ** P < 0.01 calculated using two-tailed Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: In Vitro, Concentration Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: ERAP1 inhibition impairs Hh-dependent tumor growth in vivo. a – f NSG mice were grafted with spontaneous primary MB from Math1-cre/Ptc C/C mice. Tumor masses (150 mm 3 ) were intratumorally injected with Leu-SH. a Tumor growth was monitored. b Representative flank allograft average volumes (lower panel) and quantification of tumor explants (upper panel). c , d Ki67, NeuN, and cleaved Caspase-3 (Cl.Casp-3) immunohistochemical stainings of allograft tumor samples. d Quantification of immunohistochemical stainings shown in c . Scale bar 100 μm. e mRNA and f protein expression levels of Hh targets from tumors assayed in b . g Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor in NSG mice after i.p. injection of Leu-SH. Scale bars, 500 μm and 200 μm (upper and lower panels, respectively). h Representative average volume of orthotopic tumor. i – j NSG mice were grafted with spontaneous primary MB from Math1-cre/Ptc C/C mice genetically silenced for ERAP1 expression. i Representative images of mice and the explanted tumor masses. j Quantification of the flank allograft average tumor volume. ERAP1 protein expression is shown below. In f , j , actin was used as loading control. k H&E and representative Masson’s trichrome staining of tumors. Scale bar 100 μm. l mRNA levels of the indicated Hh target genes. m Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor genetically interfered for ERAP1 before the injection in NSG mice cerebella. Scale bars, 500 and 200 μm (upper and lower panels, respectively). n – p ERAP1 accelerates Hh-MB formation. n Tumor volume of mice subcutaneously transplanted with GCPs from tumor-prone Math1-cre/Ptc C/C animals overexpressing ERAP1. o Representative flank allograft average volumes (lower panel) and quantification of the explanted tumor masses (upper panel). p mRNA expression of Hh target genes from the tumor masses assayed in o . q Survival curves of Math-cre/Ptc C/C mice treated with Leu-SH or vehicle. Results in e , l , p were normalized to endogenous GAPDH and HPRT controls and expressed as in Fig. . All data represent the mean of three independent experiments. Mean ± S.D. of tumor ( n = 6) for each treatment. * P < 0.05, ** P < 0.01, *** P < 0.001 calculated by two-sided Student’s t -test

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Inhibition, In Vivo, Injection, Immunohistochemical staining, Expressing, Derivative Assay, Staining

Journal: Nature Communications

Article Title: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP

doi: 10.1038/s41467-019-11093-0

Figure Lengend Snippet: A representative model showing the role of ERAP1 in Hh-dependent tumorigenesis. ERAP1 promotes ubiquitylation and proteasomal degradation of βTrCP by sequestering USP47. This event leads to increase of Gli1 and Gli2 protein levels and decrease of Gli3R, thus triggering the Hh pathway and favoring cell growth and tumorigenesis. In the absence of ERAP1, USP47 binds and stabilizes βTrCP, which, in turn, promotes ubiquitylation and proteasomal degradation of Gli1 and Gli2, and ubiquitylation and proteolytic cleavage of Gli3 into the repressor form Gli3R. These events lead to the repression of the Hh pathway and inhibition of cell proliferation and tumor growth

Article Snippet: Oro. pCMV6-XL5-ERAP1 (SC311137) was purchased from Origene (Rockville, MD, USA). shCTRL (SHC002) and shERAP1 (TRCN0000031119, TRCN0000031121, TRCN0000060542) in pLKO.1 plasmids were purchased from Sigma-Aldrich.

Techniques: Inhibition

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Association between ERAP1 gene polymorphisms and ankylosing spondylitis susceptibility in Han population

doi:

Figure Lengend Snippet: Primer sequences of ERAP1 rs27434 and rs7711564 polymorphisms

Article Snippet: Taqman probes (ABI company) for rs27434 and rs7711564 polymorphisms were respectively C_794791_10 and C_3056830_10.

Techniques: Sequencing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Association between ERAP1 gene polymorphisms and ankylosing spondylitis susceptibility in Han population

doi:

Figure Lengend Snippet: Comparison of genotype and allele frequencies of ERAP1 rs27434 and rs7711564

Article Snippet: Taqman probes (ABI company) for rs27434 and rs7711564 polymorphisms were respectively C_794791_10 and C_3056830_10.

Techniques: Comparison

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Association between ERAP1 gene polymorphisms and ankylosing spondylitis susceptibility in Han population

doi:

Figure Lengend Snippet: Primer sequences of ERAP1 rs27434 and rs7711564 polymorphisms

Article Snippet: Taqman probes (ABI company) for rs27434 and rs7711564 polymorphisms were respectively C_794791_10 and C_3056830_10.

Techniques: Sequencing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Association between ERAP1 gene polymorphisms and ankylosing spondylitis susceptibility in Han population

doi:

Figure Lengend Snippet: Comparison of genotype and allele frequencies of ERAP1 rs27434 and rs7711564

Article Snippet: Taqman probes (ABI company) for rs27434 and rs7711564 polymorphisms were respectively C_794791_10 and C_3056830_10.

Techniques: Comparison

Journal: International Journal of Molecular Sciences

Article Title: ERAP/HLA-C and KIR Genetic Profile in Couples with Recurrent Implantation Failure

doi: 10.3390/ijms232012518

Figure Lengend Snippet: Summarized effect of ERAP , HLA-C , KIR polymorphisms on susceptibility to infertility and recurrent implantation failure.

Article Snippet: rs27044 , C > G , Gln730Glu , ERAP1 , 5q15 , Enzymatic activity, substrate length preference [ ] , C__3056870_10.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: ERAP/HLA-C and KIR Genetic Profile in Couples with Recurrent Implantation Failure

doi: 10.3390/ijms232012518

Figure Lengend Snippet: ERAP1 and ERAP2 studied polymorphisms.

Article Snippet: rs27044 , C > G , Gln730Glu , ERAP1 , 5q15 , Enzymatic activity, substrate length preference [ ] , C__3056870_10.

Techniques: Activity Assay, Expressing, Functional Assay

Journal: bioRxiv

Article Title: ERAP1 Shows Distinct Regulatory Mechanisms on Blood Pressure Modulation Between Males and Females

doi: 10.1101/2023.06.07.544152

Figure Lengend Snippet: A , ERAP1 mRNA levels in the adrenal tissue detected by RT- q PCR; wt: n =14, Het: n =16. B, Western blots and ( C ) representative optical densitometry of ERAP1 in the adrenals, normalized to GAPDH; wt: n =5, Het: n =5 independent experiments. D, Immunofluorescence labeling of ERAP1 expression in the adrenal cortex of males. Nuclei counterstained with DAPI. Scale bars=50 µm.; n =5 independent experiments. E, Quantification of ERAP1 from the cortex of adrenals in the same male mice as D ; n =5 independent experiments. F, Immunofluorescence labeling of ERAP1 expression in the aortas from females. Nuclei counterstained with DAPI. Scale bars=100 µm; n =6 independent experiments. G, Quantification of ERAP1 in the aortas from same female mice as F ; n =6 independent experiments. ( H ) Systolic BP (blood pressure), ( I ) diastolic BP, and ( J ) HR (heart rate) were measured in mice by tail-cuff plethysmography after 4 weeks of liberal-salt diets; wt: n =37, Het: n =55. K, SSBP (SSBP = systolic BP on a liberal salt diet -systolic BP on a restricted salt diet) in human subjects <51 years; Non-carriers (NC): n =25, Carriers (C): n =25. The absolute concentration of Ang II in ( L ) the hearts, ( M ) adrenals and ( N ) aortas; wt: n =20, Het: n =19. P values are determined by Student t test, * P <0.05, ** P <0.01, and *** P <0.001. Data in the dot plots represent the mean ± SEM. wt: wild type; Het: ERAP1 +/- ; Ang II: angiotensin II; BP: blood pressure; HR: heart rate; BPM: beats per minute.

Article Snippet: Membranes were blocked in 5% nonfat dry milk (Bio-Rad) for 1 hour at room temperature, incubated with primary antibodies to ERAP1 (1:1000, Bioworld, BS2155) and GAPDH (1:2000, Santa Cruz Biotechnology Inc., sc-47724) at 4°C overnight, and then stained with HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology Inc. sc-2004) for 1 hour at room temperature.

Techniques: Western Blot, Immunofluorescence, Labeling, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: ERAP1 Shows Distinct Regulatory Mechanisms on Blood Pressure Modulation Between Males and Females

doi: 10.1101/2023.06.07.544152

Figure Lengend Snippet: A , ERAP1 mRNA levels in the adrenal tissues detected by RT- q PCR; wt M: n =8, Het M: n =7, wt F: n =6, Het F: n =8. B, Western blots and ( C ) representative optical densitometry of ERAP1 in the adrenals, normalized to GAPDH; n =6 independent experiments. ( D ) Systolic BP (blood pressure), ( E ) diastolic BP, and ( F ) HR (heart rate) collected from mice with 4 weeks of liberal-salt diets by tail-cuff plethysmography; wt M: n =19, Het M: n =26, wt F: n =18, Het F: n =29. G , SSBP (SSBP = systolic BP on a liberal salt diet - systolic BP on a restricted salt diet) in human subjects <51 years. Non-carriers M: n =24, Carriers M: n =28, Non-carriers F: n =26, Carriers F: n =22. H, 1.6% Na + intake within 24 hours; wt M: n =13, Het M: n =15, wt F: n =12, Het F: n =18. The absolute concentration of Ang II in ( I ) the adrenals, and ( J ) the aortas; wt M: n =10, Het M: n =10, wt F: n =9, Het F: n =9. Data in the dot plots represent the mean ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 by 1-way ANOVA for multiple groups. wt: wild type; Het: ERAP1 +/- ; M: male; F: female; Ang II: angiotensin II; BP: blood pressure; HR: heart rate; BPM: beats per minute.

Article Snippet: Membranes were blocked in 5% nonfat dry milk (Bio-Rad) for 1 hour at room temperature, incubated with primary antibodies to ERAP1 (1:1000, Bioworld, BS2155) and GAPDH (1:2000, Santa Cruz Biotechnology Inc., sc-47724) at 4°C overnight, and then stained with HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology Inc. sc-2004) for 1 hour at room temperature.

Techniques: Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: ERAP1 Shows Distinct Regulatory Mechanisms on Blood Pressure Modulation Between Males and Females

doi: 10.1101/2023.06.07.544152

Figure Lengend Snippet: A , Adrenal and body weights collected from Het and wt mice at the time of scarification; wt M: n =17, Het M: n =19, wt F: n =20, Het F: n =20. B, Serum Aldo level detected by Enzyme-Linked Immunosorbent Assay (ELISA) kits; wt M: n =8, Het M: n =9, wt F: n =11, Het F: n =7, and ( C ) Aldo levels of 24 hours urines which were collected by metabolic cages from mice on a liberal-salt diet were measured by ELISA immunoassays; wt M: n =8, Het M: n =14, wt F: n =9, Het F: n =18. D, Macro-morphology of the adrenals from 20-week-old mice was detected by H&E staining. Scale bars=200 µm. n =5 independent experiments. E, Micro-morphology of adrenals from 20-week-old mice was detected by H&E staining. Scale bars=50 µm. n =5 independent experiments. F, IHC staining with antibodies (green) against ERAP1 in the adrenal cortex and quantification of ERAP1 in ( G ) ZG cells and ( H ) ZF cells. Scale bars =50 µm. Nuclei counterstained with DAPI; n =5 independent experiments. I, IHC staining with antibodies (green) against CYP11B2 in the adrenal cortex and quantification of CYP11B2 in ( J ) ZG cells and ( K ) ZF cells. Scale bars =50 µm. Nuclei counterstained with DAPI; n =5 independent experiments. Data in the dot plots represent the mean ± SEM. * P <0.05 and *** P <0.001 by 1-way ANOVA for multiple groups. wt: wild type; Het: ERAP1 +/- ; M: male; F: female; ZG: zona glomerulosa; ZF: zona fasciculata; Aldo: aldosterone; IHC: immunochemistry.

Article Snippet: Membranes were blocked in 5% nonfat dry milk (Bio-Rad) for 1 hour at room temperature, incubated with primary antibodies to ERAP1 (1:1000, Bioworld, BS2155) and GAPDH (1:2000, Santa Cruz Biotechnology Inc., sc-47724) at 4°C overnight, and then stained with HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology Inc. sc-2004) for 1 hour at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: ERAP1 Shows Distinct Regulatory Mechanisms on Blood Pressure Modulation Between Males and Females

doi: 10.1101/2023.06.07.544152

Figure Lengend Snippet: A , Volcano plot of DE (differential expression) genes after ERAP1 knockdown in males. B, Heat map of batch effect by ERAP1 deletion in males. C, Biological processes of significant DE genes in the same male mice as A and B . D, Volcano plot of DE genes after ERAP1 knockdown in females. E, Heat map of batch effect by ERAP1 deletion in females. F, Biological processes of significant DE genes in the same female mice as D and E . wt M: n =3, Het M: n =3, wt F: n =3, Het F: n =3. wt: wild type; Het or heterozygous: ERAP1 +/- ; M: male; F: female.

Article Snippet: Membranes were blocked in 5% nonfat dry milk (Bio-Rad) for 1 hour at room temperature, incubated with primary antibodies to ERAP1 (1:1000, Bioworld, BS2155) and GAPDH (1:2000, Santa Cruz Biotechnology Inc., sc-47724) at 4°C overnight, and then stained with HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology Inc. sc-2004) for 1 hour at room temperature.

Techniques: Expressing

Journal: bioRxiv

Article Title: ERAP1 Shows Distinct Regulatory Mechanisms on Blood Pressure Modulation Between Males and Females

doi: 10.1101/2023.06.07.544152

Figure Lengend Snippet: A , Aorta and body weights collected from Het and wt mice when they were sacrificed; wt M: n =13, Het M: n =12, wt F: n =13, Het F: n =13. B, RPF value transferred from PAH concentration of plasma in ERAP1 +/- mice and wt littermates; wt M: n =6, Het M: n =9, wt F: n =5, Het F: n =9. C, Morphology of the aortas detected by H&E staining; Scale bars=200 µm. n =6 independent experiments. D, IHC staining with antibodies (green) against ERAP1 in the aortas. Nuclei counterstained with DAPI. Scale bars =100 µm; n =6 independent experiments. E, IHC staining with antibodies (green) against CD31 in the aortas. Nuclei counterstained with DAPI. Scale bars =100 µm; n =6 independent experiments. F, Quantification of ERAP1 expression in endothelial cells from the same mice with D ; n =6 independent experiments. G, Quantification of CD31 in endothelial cells from the same mice with E ; n =6 independent experiments. Data in the dot plots represent the mean ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 by 1-way ANOVA for multiple groups. wt: wild type; Het: ERAP1 +/- ; M: male; F: female; RPF: renal plasma flow; PAH: N-(4-aminobenzoyl)glycin; IHC: immunochemistry.

Article Snippet: Membranes were blocked in 5% nonfat dry milk (Bio-Rad) for 1 hour at room temperature, incubated with primary antibodies to ERAP1 (1:1000, Bioworld, BS2155) and GAPDH (1:2000, Santa Cruz Biotechnology Inc., sc-47724) at 4°C overnight, and then stained with HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology Inc. sc-2004) for 1 hour at room temperature.

Techniques: Concentration Assay, Staining, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: ERAP1 Shows Distinct Regulatory Mechanisms on Blood Pressure Modulation Between Males and Females

doi: 10.1101/2023.06.07.544152

Figure Lengend Snippet: A , Summary of experiment design in different three groups (Control, Enalapril and Amlodipine). BP and HR (heart rate) track in the same mouse by tail-cuff measurements before and after given the oral administration of ( B )Enalapril and ( C ) Amlodipine; Enalapril: n =31; Amlodipine: n =31. ( D ) Systolic BP, ( E ) diastolic BP, and ( F ) HR were measured by tail-cuff plethysmography in Control, Enalapril and Amlodipine groups. Δ Systolic BP = systolic BP collected from mice before medication - systolic BP collected from mice after medication. Δ Diastolic BP = diastolic BP collected from mice before medication - diastolic BP collected from mice after medication. Control: n =31; Enalapril: n =31; Amlodipine: n =31; wt M: n =6, Het M: n =9, wt F: n =6, Het F: n =10; wt: n =12, Het: n =19. G, 1.6% Na + intake within 24 hours in Control group, Enalapril group and Amlodipine group; Control: n =31, Enalapril: n =31, Amlodipine: n =31. Data in the dot plots represent the mean ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 by 1-way ANOVA for multiple groups. wt: wild type; Het: ERAP1 +/- ; M: male; F: female; BP: blood pressure; HR: heart rate; BPM: beats per minute; Control: mice with liberal salt diets (1.6% Na + ); Enalapril: mice with liberal salt diets containing Enalapril (15 mg/kg/day) for 14 days; Amlodipine: mice with liberal salt diets containing Amlodipine (7.5 mg/kg/day) for 14 days.

Article Snippet: Membranes were blocked in 5% nonfat dry milk (Bio-Rad) for 1 hour at room temperature, incubated with primary antibodies to ERAP1 (1:1000, Bioworld, BS2155) and GAPDH (1:2000, Santa Cruz Biotechnology Inc., sc-47724) at 4°C overnight, and then stained with HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology Inc. sc-2004) for 1 hour at room temperature.

Techniques:

Journal: bioRxiv

Article Title: ERAP1 Shows Distinct Regulatory Mechanisms on Blood Pressure Modulation Between Males and Females

doi: 10.1101/2023.06.07.544152

Figure Lengend Snippet: A , Serum Aldo collected from mice fed with liberal salts (Control), or Enalapril, or Amlodipine was measured by Enzyme-Linked Immunosorbent Assay (ELISA) kits; Control: n =36; Enalapril: n =36; Amlodipine: n =36; wt M: n =9, Het M: n =9, wt F: n =11, Het F: n =7. B, RPF measured from PAH concentration of plasma in mice with liberal salts (Control) or Enalapril or Amlodipine administration. Control: n =31; Enalapril: n =31; Amlodipine: n =31; wt M: n =6, Het M: n =9, wt F: n =6, Het F: n =10. Data in the dot plots represent the mean ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 by 1-way ANOVA for multiple groups. wt: wild type; Het: ERAP1 +/- ; M: male; F: female; Aldo: aldosterone; RPF: renal plasma flow; PAH: N-(4-aminobenzoyl)glycin; Control: mice with liberal salt diets (1.6% Na + ); Enalapril: mice with liberal salt diets containing Enalapril (15 mg/kg/day) for 14 days; Amlodipine: mice with liberal salt diets containing Amlodipine for 14 days (7.5 mg/kg/day).

Article Snippet: Membranes were blocked in 5% nonfat dry milk (Bio-Rad) for 1 hour at room temperature, incubated with primary antibodies to ERAP1 (1:1000, Bioworld, BS2155) and GAPDH (1:2000, Santa Cruz Biotechnology Inc., sc-47724) at 4°C overnight, and then stained with HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology Inc. sc-2004) for 1 hour at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Molecular immunology

Article Title: ERAP1 Reduces Accumulation of Aberrant and Disulfide-Linked Forms of HLA-B27 on the Cell Surface

doi: 10.1016/j.molimm.2016.04.002

Figure Lengend Snippet: (A) Whole cell extracts from U937.B27 cells stably expressing ERAP1 or scrambled (Scram) shRNA were subjected to SDS-PAGE and blotted for ERAP1 and GAPDH as a loading control. (B) ERAP1 shRNA knockdown and control (Scram) U937.B27 cells were stained with the antibodies indicated, and analyzed by flow cytometry. Relative expression is mean fluorescence intensity for ERAP1 knockdown cells compared to cells transfected with scrambled shRNA (set to 1). Data are from 2–4 independent experiments done in triplicate. (C,D) Cell surface HLA class I was IPd with W6/32 (C) or HC10 (D), treated with N-glycanase, and quantitated by IEF and Western blotting. Representative blots (left panels) and quantitative results (right panels) are shown. Longer exposures are required to better visualize the B27 band in (D). Since this results in overexposure of the B51 band, we show only the shorter exposure here. A longer exposure is shown in Supplemental Figure S1C. Relative expression is the mean (+/−SEM) of triplicate samples, with expression in scrambled shRNA cells normalized to 1. * P < 0.05; **P < 0.01.

Article Snippet: U937.B27 cells were infected with retrovirus encoding ERAP1 shRNA or scrambled control shRNA (Origene, Rockville, MD), and selected with puromycin and for loss of ERAP1 expression by immunoblotting.

Techniques: Stable Transfection, Expressing, shRNA, SDS Page, Staining, Flow Cytometry, Fluorescence, Transfection, Western Blot

Journal: Molecular immunology

Article Title: ERAP1 Reduces Accumulation of Aberrant and Disulfide-Linked Forms of HLA-B27 on the Cell Surface

doi: 10.1016/j.molimm.2016.04.002

Figure Lengend Snippet: (A) Cell surface complexes were immunoprecipitated from ERAP1 knockdown and control (Scram) U937.B27 cells with HC10 and analyzed by SDS-PAGE under non-reducing conditions. Representative (left) and quantitative (right) results from triplicates are shown. High molecular weight complexes (HMW) migrate in the 85–90 kDa size range and are eliminated when samples are reduced prior to electrophoresis (not shown). (B) Whole cell HC10 immunoprecipitations were performed and analyzed as described in (A). Quantitative results are from three experiments with duplicate gels. * P < 0.05

Article Snippet: U937.B27 cells were infected with retrovirus encoding ERAP1 shRNA or scrambled control shRNA (Origene, Rockville, MD), and selected with puromycin and for loss of ERAP1 expression by immunoblotting.

Techniques: Immunoprecipitation, SDS Page, Molecular Weight, Electrophoresis

Journal: Molecular immunology

Article Title: ERAP1 Reduces Accumulation of Aberrant and Disulfide-Linked Forms of HLA-B27 on the Cell Surface

doi: 10.1016/j.molimm.2016.04.002

Figure Lengend Snippet: ERAP1 knockdown and scrambled shRNA (Scram) U937.B27 cells were treated with IFN-γ (100 ng/ml) or PBS for 24 hours. (A) Whole cell extracts were subjected to SDS-PAGE and blotted for ERAP1 and GAPDH as a loading control. (B) Cells were stained with the antibodies indicated, and analyzed by flow cytometry. For each antibody, expression in PBS-treated scrambled shRNA cells was normalized to 1. Data are expressed as mean +/−SEM from 2–4 experiments done in duplicate or triplicate. (C,D) Cell surface HLA class I was IPd with W6/32 (C) or HC10 (D) and quantitated by IEF and Western blotting as described in the legend to Figure 2 (C,D). Representative blots (left panels) and quantitative results (right panels) are shown. The representative images for no IFNγ treatment are the same as those shown in Figure 2C,D, and are included here to allow comparison with +IFNγ. Expression is the mean (+/−SEM) of triplicate samples, relative to PBS-treated scrambled shRNA cells. * P < 0.05; **P < 0.01.

Article Snippet: U937.B27 cells were infected with retrovirus encoding ERAP1 shRNA or scrambled control shRNA (Origene, Rockville, MD), and selected with puromycin and for loss of ERAP1 expression by immunoblotting.

Techniques: shRNA, SDS Page, Staining, Flow Cytometry, Expressing, Western Blot

Journal: Molecular immunology

Article Title: ERAP1 Reduces Accumulation of Aberrant and Disulfide-Linked Forms of HLA-B27 on the Cell Surface

doi: 10.1016/j.molimm.2016.04.002

Figure Lengend Snippet: ERAP1 knockdown and scrambled shRNA (Scram) U937.B27 cells were treated with IFN-γ (100 ng/ml) or PBS for 24 hours. (A) Cell surface complexes were immunoprecipitated with HC10 and analyzed by SDS-PAGE under non-reducing conditions. Representative (left) and quantitative (right) results from triplicates are shown. High molecular weight complexes (HMW) migrate in the 85–90 kDa size range and are eliminated when samples are reduced prior to electrophoresis (not shown). (B) Whole cell lysate HC10 immunoprecipitations were performed and analyzed as described in (A). Quantitative results are from three experiments with duplicate gels. * P < 0.05

Article Snippet: U937.B27 cells were infected with retrovirus encoding ERAP1 shRNA or scrambled control shRNA (Origene, Rockville, MD), and selected with puromycin and for loss of ERAP1 expression by immunoblotting.

Techniques: shRNA, Immunoprecipitation, SDS Page, Molecular Weight, Electrophoresis

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: The association of HLA-C and ERAP1 polymorphisms in early and late onset psoriasis and psoriatic arthritis patients of Hungary

doi: 10.5114/ada.2021.104277

Figure Lengend Snippet: ERAP1 and HLA-Cw*0602 gene SNPs

Article Snippet: 5 , ERAP1 , rs17482078 , C___3056871_10 , C/T , Arg725Gln.

Techniques:

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: The association of HLA-C and ERAP1 polymorphisms in early and late onset psoriasis and psoriatic arthritis patients of Hungary

doi: 10.5114/ada.2021.104277

Figure Lengend Snippet: Distribution of ERAP1 and HLA-Cw SNP genotypes in healthy controls and psoriasis patients

Article Snippet: 5 , ERAP1 , rs17482078 , C___3056871_10 , C/T , Arg725Gln.

Techniques:

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: The association of HLA-C and ERAP1 polymorphisms in early and late onset psoriasis and psoriatic arthritis patients of Hungary

doi: 10.5114/ada.2021.104277

Figure Lengend Snippet: Linkage disequilibrium (LD) for the ERAP1 SNPs in the study population

Article Snippet: 5 , ERAP1 , rs17482078 , C___3056871_10 , C/T , Arg725Gln.

Techniques:

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: The association of HLA-C and ERAP1 polymorphisms in early and late onset psoriasis and psoriatic arthritis patients of Hungary

doi: 10.5114/ada.2021.104277

Figure Lengend Snippet: Distribution of ERAP1 haplotypes in healthy controls and psoriasis patients

Article Snippet: 5 , ERAP1 , rs17482078 , C___3056871_10 , C/T , Arg725Gln.

Techniques:

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: The association of HLA-C and ERAP1 polymorphisms in early and late onset psoriasis and psoriatic arthritis patients of Hungary

doi: 10.5114/ada.2021.104277

Figure Lengend Snippet: HLA-C and ERAP1 interactions in psoriasis and psoriatic arthritis

Article Snippet: 5 , ERAP1 , rs17482078 , C___3056871_10 , C/T , Arg725Gln.

Techniques: