Journal: Frontiers in Immunology

Article Title: RUVBL1/2 Complex Regulates Pro-Inflammatory Responses in Macrophages via Regulating Histone H3K4 Trimethylation

doi: 10.3389/fimmu.2021.679184

Figure Lengend Snippet: RUVBL2 is essential for Nos2 gene expression and bactericidal activity of macrophages (A) Expression of Ruvbl2 in RAW264.7 cells. Cells were transfected with SMARTpool siRNA (SP-siRuvbl2), individual siRNA (siRuvbl2#1, siRuvbl2#2), or control siRNA (siControl) for 48 hours. Left, RT-PCR analysis of relative Ruvbl2 mRNA expression in response to different siRNA transfections. Right, Western blot analysis of RUVBL2 expression in cells transfected with siControl and SP-siRuvbl2 (upper panel), and with siControl, siRuvbl2#1 and siRuvbl2#2 respectively (lower panel). (B) Relative Nos2 mRNA expression in RAW 264.7 cells transfected with the corresponding siRNAs in the presence or absence of LPS (10 ng/ml) for 24 hours. (C) Level of nitrite in culture medium of RAW 264.7 cells transfected with the corresponding siRNAs in the presence or absence of LPS (10 ng/ml) for 24 hours. (D) Left, expression of Ruvbl1 gene in RAW 264.7 cells upon transfection with control siRNA (siRuvbl1) or siRNA against Ruvbl1 (siRuvbl1). Middle, level of Nos2 expression in RAW 264.7 cells transfected with the corresponding siRNAs in the presence or absence of LPS (10 ng/ml) for 24 hours. Right, level of nitrite in culture medium of RAW 264.7 cells transfected with the corresponding siRNAs in the presence or absence of LPS (10 ng/ml) for 24 hours. (E) RAW 264.7 cells transfected with siControl or SP-siRuvbl2 were infected with E.coli . The level of bacterial load (left) and nitrite levels in the corresponding condition medium after 24 h (right) were determined. (F) Western blotting analysis showing the expression of RUVBL2 in whole cell lysate (Upper panel), and subcellular fractions (Lower panel), upon LPS treatment for different time points. Data from (A–E) were obtained from three independent experiments and presented in mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA with Bonferroni’s multiple comparison test as post-test in (A–C) and by unpaired t-test in (D, E) .

Article Snippet: The following assays were used: Ruvbl2 (Mm00600028_m1), Ruvbl1 (Mm04203863_g1), Actb (Mm02619580_g1), Ms4a7 (Mm01197655_m1), Marcks (Mm02524303_s1), Il6 (Mm00446190_m1), Csf3 (Mm00438334_m1), Fpr2 (Mm00484464_s1), Ccnd2 (Mm00438070_m1), Vegfc (Mm00437310_m1), Csf2 (Mm01290062_m1), Il1f6 (Mm00457645_m1), Cxcl2 (Mm00436450_m1), Siglec1 (Mm00488332_m1), Lcn2 (Mm01324470_m1), Ccr3 (Mm01216172_m1), Acod1 (Mm01224532_m1), Ccl6 (Mm01302419_m1), Nos2 (Mm00440502_m1), Tnfrsf1b (Mm00441889_m1), Ccl7 (Mm00443113_m1), Il1b (Mm00434228_m1), Ccl2 (Mm00441242_m1), Socs3 (Mm00545913_s1), Ccl3 (Mm00441259_g1), Ccl4 (Mm00443111_m1), Car13 (Mm01291526_m1), Pkm (Mm00834102_gH), Ccl5 (Mm01302427_m1), Pck1 (Mm01247058_m1), Il33 (Mm00505403_m1), Mx1 (Mm00487796_m1), Serpinb3b (Mm03032256_uH), Tnf (Mm00443258_m1), Ptgs2 (Mm00478374_m1).

Techniques: Gene Expression, Activity Assay, Expressing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Comparison

Journal: Frontiers in Immunology

Article Title: RUVBL1/2 Complex Regulates Pro-Inflammatory Responses in Macrophages via Regulating Histone H3K4 Trimethylation

doi: 10.3389/fimmu.2021.679184

Figure Lengend Snippet: RUVBL2 is essential for pro-inflammatory gene expressions. (A) Heatmap of pro-inflammatory gene expression using real-time PCR analysis at 4 and 24 hrs after LPS induction of RAW 264.7 macrophages transfected with siControl or SP-siRuvbl2. (B) Level of TNF-α, IFNγ, IL-6, IL-1β, and GM-CSF in culture medium of RAW 264.7 macrophages transfected with siControl or siRuvbl2, in the presence of LPS (10 ng/ml). (C) Kinetics of expression of representative primary response genes in LPS (10 ng/ml)-induced RAW 264.7 macrophages transfected with siControl or SP-siRuvbl2. (D) Kinetics of representative secondary response genes expression in siControl and SP-siRuvbl2 transfected cells in response to LPS (10 ng/ml). Data from (A–D) are obtained from three independent experiments and presented in mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, by unpaired t-test in (B) and by two-way ANOVA with Bonferroni’s multiple comparison test as post-test in (C, D) .

Article Snippet: The following assays were used: Ruvbl2 (Mm00600028_m1), Ruvbl1 (Mm04203863_g1), Actb (Mm02619580_g1), Ms4a7 (Mm01197655_m1), Marcks (Mm02524303_s1), Il6 (Mm00446190_m1), Csf3 (Mm00438334_m1), Fpr2 (Mm00484464_s1), Ccnd2 (Mm00438070_m1), Vegfc (Mm00437310_m1), Csf2 (Mm01290062_m1), Il1f6 (Mm00457645_m1), Cxcl2 (Mm00436450_m1), Siglec1 (Mm00488332_m1), Lcn2 (Mm01324470_m1), Ccr3 (Mm01216172_m1), Acod1 (Mm01224532_m1), Ccl6 (Mm01302419_m1), Nos2 (Mm00440502_m1), Tnfrsf1b (Mm00441889_m1), Ccl7 (Mm00443113_m1), Il1b (Mm00434228_m1), Ccl2 (Mm00441242_m1), Socs3 (Mm00545913_s1), Ccl3 (Mm00441259_g1), Ccl4 (Mm00443111_m1), Car13 (Mm01291526_m1), Pkm (Mm00834102_gH), Ccl5 (Mm01302427_m1), Pck1 (Mm01247058_m1), Il33 (Mm00505403_m1), Mx1 (Mm00487796_m1), Serpinb3b (Mm03032256_uH), Tnf (Mm00443258_m1), Ptgs2 (Mm00478374_m1).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Transfection, Expressing, Comparison

Journal: eLife

Article Title: EHMT2 methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation

doi: 10.7554/eLife.57648

Figure Lengend Snippet: ( A ) Western blot demonstrating expression of HIF1-α and β-catenin in A549 lysates immunoprecipitated with RUVBL2 antibody. Cells were cultured under hypoxic conditions (1% O 2 ) vs control (ambient O 2 ). ( B ) Western blot demonstrating co-immunoprecipitation of RUVBL2 in A549 lysates co-immunoprecipitated with β-catenin antibody. Cells were treated with EHMT2 inhibitor vs control. ( C ) Proximity ligation assay in EHMT2i-treated vs vehicle-treated tumor-propagating cells (TPCs) (red, RUVBL2-β-catenin proximity ligation; blue, DAPI counterstain). Insets show a magnification of the red signal in nuclei of vehicle-treated TPCs (scale 10 μm). ( D ) Quantification of normalized nuclei with a positive signal (n=2; mean ± SEM, two-tailed paired t-test, **p<0.05). ( E ) Cytoplasmic (cyt), nuclear (nuc) and chromatin (chro) subcellular fractionation of EHMT2i-treated (as indicated) tumorspheres compared to control. Histone H4 and Tubulin are loading controls of chromatin and cytoplasmic fractions, respectively. ( F ) Chromatin immunoprecipitation using RUVBL2 antibody followed by qPCR (ChIP-qPCR) of areas flanking a Tcf4 binding sites in promoters of the alveolar type 2 (AT2) genes Etv5 , Slc34a2 and Lamp3 (n=2 mean ± SEM; Etv5 , Slc34a2 , two-tailed paired t-test, *p<0.05). ( G ) Representative qPCR of Tcf4 binding motif within promoters of AT2 genes. Figure 5—source data 1. Western blot for showing G9a (EHMT2) expression in subcellular fractionation of EHMT2i-treated tumorspheres compared to control. Figure 5—source data 2. Western blot for showing TUBULIN expression in middle membrane (upper band) in subcellular fractionation of EHMT2i-treated tumorspheres compared to control. Figure 5—source data 3. Western blot for showing histone H4 expression in the bottom membrane and beta catenin expression (upper membrane) in subcellular fractionation of EHMT2i-treated tumorspheres compared to control.

Article Snippet: Antibody , (rabbit polyclonal); RUVBL2 , Bethyl Laboratories , A5302-537A , Immunoprecipitation.

Techniques: Western Blot, Expressing, Immunoprecipitation, Cell Culture, Control, Proximity Ligation Assay, Ligation, Two Tailed Test, Fractionation, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Membrane

Journal: eLife

Article Title: EHMT2 methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation

doi: 10.7554/eLife.57648

Figure Lengend Snippet:

Article Snippet: Antibody , (rabbit polyclonal); RUVBL2 , Bethyl Laboratories , A5302-537A , Immunoprecipitation.

Techniques: Control, Western Blot, Immunoprecipitation, Concentration Assay, Transfection, Construct, Software

Journal: Journal of Biological Chemistry

Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

doi: 10.1074/jbc.ra118.006290

Figure Lengend Snippet: Figure 4. Nrf1 interacts with the TIP60 complex. (A) Wild-type (WT) and Nrf1-/- NIH-3T3 cell lines were treated with 200 nM carfilzomib (CFZ) for 8 hours. The cells were then subjected to chromatin immunoprecipitation (ChIP) with one of IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for antioxidant response element (ARE)- containing promoter regions of proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote SD (n=3). (B) HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated or not with 200 nM CFZ for 8 hours. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times and a representative blot is shown.

Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: Advanced Science

Article Title: Epigenetic Control of Translation Checkpoint and Tumor Progression via RUVBL1‐EEF1A1 Axis

doi: 10.1002/advs.202206584

Figure Lengend Snippet: RUVBL1 controls MYC chromatin localization and transactivation activity in EwS. A) RNAseq and GSEA analyses showing changes in expression of the MYC upregulated target gene set in sgCtrl and sgRUVBL1 transduced (day 5) A673‐Cas9 cells (two independent sgRNA sequences per group). (Right) Each dot indicates one gene set from the GSEA Molecular Signature Database (MSigDB; total 238 gene sets from the Hallmark and Oncogenic Signature [C6] collections). NES: Normalized enrichment score. B) Growth competition assay of sgCtrl (gray lines; n = 2 independent sgRNA sequences) and sgMYC (purple lines; n = 5 independent sgRNA sequences) in A673‐Cas9 cells. C) Cell cycle monitored by EdU incorporation, and D) cellular apoptosis detected by active caspase 3 + /DAPI − in A673‐Cas9 cells transduced with sgCtrl and sgMYC ( n = 3 for each group). E) Western blot of RUVBL1, MYC, histone H4, and GAPDH in A673‐Cas9 cells transduced with sgCtrl and sgRUVBL1 (two independent sgRNA sequences per group). F) Co‐IP of RUVBL1 (flag‐tagged) with RUVBL2 and MYC in HEK293 cells. G) Meta plots (top) and heatmaps (bottom) showing ChIP‐seq signal of MYC at TSSs ± 3 kb regions for all genes in A673‐Cas9 cells transduced with sgCtrl and sgRUVBL1. H) Profiles of MYC ChIP‐seq and (I) ChIP‐qPCR at RUVBL1 locus in A673‐Cas9 cells transduced with sgCtrl and sgRUVBL1 ( n = 3 for each group). J) RT‐qPCR of RUVBL1 mRNA in A673‐Cas9 cells transduced with sgCtrl and sgMYC ( n = 3 for each group). K) Western blot of MYC, RUVBL1, and GAPDH in A673‐Cas9 cells transduced with sgMYC (two independent sgRNA sequences per group). L) Model of a feed‐forward network between RUVBL1 and MYC in EwS. Data are represented as mean ± SEM. * P < 0.01 compared to sgCtrl by two‐sided Student's t ‐test.

Article Snippet: PVDF membranes were immersed in 5% nonfat milk then incubated at 4 °C overnight with primary antibodies against RUVBL1 (HPA019947, Sigma; 1:1000), RUVBL2 (12668S, Cell Signaling Technology; 1:1000), MYC (13987S, Cell Signaling Technology; 1:1000), EEF1A1 (PA5‐17213, Thermo Fisher; 1:1000), KAT5 (12058S, Cell Signaling Technology; 1:1000), H4K8ac (07‐328, Millipore; 1:1000), H4K12ac (61527, Active Motif; 1:1000), histone H4 (ab177840, Abcam; 1:1000), GAPDH (2118S, Cell Signaling Technology; 1:5000), flag‐tag (F7425, Millipore; 1:5000), and V5‐tag (13202S, Cell Signaling Technology; 1:1000).

Techniques: Activity Assay, Expressing, Competitive Binding Assay, Transduction, Western Blot, Co-Immunoprecipitation Assay, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Epigenetic Control of Translation Checkpoint and Tumor Progression via RUVBL1‐EEF1A1 Axis

doi: 10.1002/advs.202206584

Figure Lengend Snippet: Lysine 108 in RUVBL1 is required for the interaction between RUVBL1 and MYC. A) Schematic outline of RUVBL1 high‐density CRISPR gene body scan in A673‐Cas9 cells. B) 2D annotation of RUVBL1 CRISPR scan. The gray line indicates the smoothened model of the CRISPR scan score derived from 194 sgRNAs (dots) targeting the coding exons of RUVBL1 ( n = 3 replicates). The median CRISPR scan scores of the positive control (red line; defined as −1.0) and negative control (blue line; defined as 0.0) sgRNAs are highlighted. C) 3D annotation RUVBL1 CRISPR scan score relative to a cryo‐EM structural model of a hexamer consists of three RUVBL1 and three RUVBL2 proteins (PDB ID: 5OAF). D) Western blot showing doxycycline (DOX)‐induced expression of flag‐tagged WT‐ and K108A‐RUVBL1 in A673‐dCas9‐KRAB cells. E) Effect of WT‐ and K108A‐RUVBL1 expression on the growth competition assay of A673‐dCas9‐KRAB cells transduced with sgiCtrl and sgiRUVBL1 ( n = 3 for each group). F) Western blot of RUVBL1, H4K8ac, H4K12ac, EEF1A1, histone H4, and GAPDH in WT‐ and K108A‐RUVBL1 expressing A673‐dCas9‐KRAB cells transduced with sgiCtrl and sgiRUVBL1. G) Co‐IP of WT‐ and K108A‐RUVBL1 (flag‐tagged) with RUVBL2, KAT5, and MYC in HEK293 cells. H) Model of RUVBL1 supporting MYC chromatin binding and target gene expression. Data are represented as mean ± SEM. * P < 0.01 compared to sgCtrl by two‐sided Student's t ‐test. Source data are available for this figure: SourceData F5 B.

Article Snippet: PVDF membranes were immersed in 5% nonfat milk then incubated at 4 °C overnight with primary antibodies against RUVBL1 (HPA019947, Sigma; 1:1000), RUVBL2 (12668S, Cell Signaling Technology; 1:1000), MYC (13987S, Cell Signaling Technology; 1:1000), EEF1A1 (PA5‐17213, Thermo Fisher; 1:1000), KAT5 (12058S, Cell Signaling Technology; 1:1000), H4K8ac (07‐328, Millipore; 1:1000), H4K12ac (61527, Active Motif; 1:1000), histone H4 (ab177840, Abcam; 1:1000), GAPDH (2118S, Cell Signaling Technology; 1:5000), flag‐tag (F7425, Millipore; 1:5000), and V5‐tag (13202S, Cell Signaling Technology; 1:1000).

Techniques: CRISPR, Derivative Assay, Positive Control, Negative Control, Cryo-EM Sample Prep, Western Blot, Expressing, Competitive Binding Assay, Transduction, Co-Immunoprecipitation Assay, Binding Assay, Targeted Gene Expression

Journal: bioRxiv

Article Title: Assembly of mTORC3 involves binding of ETV7 to two separate sequences in the mTOR kinase domain

doi: 10.1101/2024.03.19.583970

Figure Lengend Snippet: A. Top drawing: schematic representation of the N- and C-terminally extended LBE fragment. The numbers indicate the number of amino acids in each of the LBE segments. Below is a FLAG immunoblot of an IP of the 120aa LBE fragment after overnight binding to ETV7 in vitro . Control (CTRL) shows the binding of human RUVBL2 to ETV7. B. Left panel, schematic representation of the different deletions of the N- and C-terminal extended LBE fragment (120 amino acids) and underneath the amino acid sequence of the LBE domain with the amino acids essential for ETV7 binding in red; Right, FLAG immunoblot of an ETV7 IP of the different LBE fragments after overnight binding to ETV7 in vitro . Lanes containing fragments that lost binding to ETV7 are boxed in red. C. FRB and LBE bind to ETV7 simultaneously. FLAG immunoblot of an ETV7 IP of LBE (120 amino acids) and Ndel-FRB fragments (ETV7: LBE: Ndel-FRB = 1:1:1, 1:1:5, 1:5:1) after overnight binding to ETV7 in vitro . D. Binding of Nde-FRB to ETV7 fragments containing the PNT domain and binding of LBE (120 amino acids) to ETV7 fragments containing the PNT and ETS domains in vitro .

Article Snippet: ETV7 protein (Cat # TP307742) and RUVBL2 (Cat # TP300933) were purchased from Origene.

Techniques: Western Blot, Binding Assay, In Vitro, Control, Sequencing

Journal: JCI Insight

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigens

doi: 10.1172/jci.insight.172449

Figure Lengend Snippet: ( A ) Proteins that were immunoprecipitated by recombinant antibody 19-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. The proteins, RUVBL1 and RUVBL2, were identified by mass spectrometry. ( B ) MiaPaca2 cells were costained with 19-3 and an antibody specific for RUVBL2. ( C ) The binding of incremental concentrations of 19-3 to RUVBL2 was measured by ELISA. ( D ) Western blot analysis of recombinant antibody 15-7 detection of cytoplasmic proteins (Cyto) and mitochondria enriched proteins (Mito) is shown. ( E ) Immunofluorescence staining of MiaPaca2 cells by 15-7 and an antibody specific for HSPD1 is shown. ( F ) The binding of incremental concentrations of 15-7 to HSPD1 was measured by ELISA. ( G ) The serum IgG titers of normal individuals ( n = 61) and PDAC patients ( n = 59) to F-actin (sera diluted 1:100), to RUVBL2 (sera diluted 1:1,000), and to HSPD1 (sera diluted 1:1,000), respectively, were measured by ELISA. Background ELISA signal is indicated by dashed line. Individual data and mean are shown. The nonparametric t test was used for comparison. Each experiment was repeated at least twice. Scale bars in B and E are 50 μm.

Article Snippet: Other antibodies and dyes used were Phalloidin (Thermo Fisher Scientific, A12379), MYH10 (Atlas Antibodies, HPA047541), cytochalasin D (Thermo Fisher Scientific, PHZ1063), Actin (Cell Signaling Technology, 3700S), RUVBL2 (Atlas Antibodies, HPA067966), RUVBL1 (Atlas Antibodies, HPA019947), HSPD1 (Atlas Antibody, HPA050025), HSPA9 (Atlas Antibody, HPA000898), COX4I1 (Atlas antibody, HPA002485), KRT19 (Abcam, ab203445), and human IgG isotype controls (BioLegend, 403502 and 403702).

Techniques: Immunoprecipitation, Recombinant, SDS Page, Silver Staining, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: Testosterone-Induced Metabolic Changes in Seminal Vesicle Epithelial cells Alter Plasma Components to Enhance Sperm Fertility

doi: 10.1101/2024.01.16.575926

Figure Lengend Snippet: (A) Tracing of oxygen consumption rate (OCR) basal and injection with 10 nM oleic acid (OA) or OA + 40 nM Etomoxir (Eto). (B) The sperm, after 1 hour of incubation with or without seminal vesicle secretions (SV) from nontreated (Ctrl) or flutamide-treated mice(Flu), were used for IVF, and the cleavaged oocytes were observed. The absolute number of oocytes collected from the oviduct and the cleavage rate are shown. (C) Western blot analysis for phosphotyrosine (P-Tyr) in capacitated spermatozoa (Ctrl; 1-hour incubation in HTF medium) and treated with SV from healthy mice or 10 nM oleic acid (OA). (D) Quantitative analysis of GLUT4 relative to α-tubulin obtained from Western blot. (E) Flow cytometric analysis of the sperm after 1 hour incubation in control medium (HTF) and medium containing SV or 10 nM OA, using fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC; to distinguish between acrosome-reacted and non-reacted cells) and propidium iodide (PI; to distinguish between dead and viable cells). (F) The percentage of viable sperm with an acrosome reaction (PI-, PNA-FITC+) was evaluated by flow cytometry in the 4th quadrant (4Q; red square). Data are mean ± SEM. At least three independent replicates. Percentage data were subjected to arcsine transformation before statistical analysis. (B) Dunnett’s test was used to analyze the cleavage rate. Different letters represent significantly different groups. (D,F) Differences between groups were assessed by one-way analysis of variance (ANOVA). When ANOVA was significant, differences among values were analyzed by Tukey’s Honest Significant Difference test for multiple comparisons.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies: anti-phosphotyrosine antibody (1:10,000; 8959; Cell Signaling), anti-GLUT4 antibody (1:100; ab33780; Abcam), anti-ACLY antibody (1:10,000; ab40793; Abcam) or anti-α/β-Tubulin antibody (1:1,000; 2148S; Cell Signaling).

Techniques: Injection, Incubation, Western Blot, Control, Flow Cytometry, Transformation Assay

Journal: Oncotarget

Article Title: Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin

doi: 10.18632/oncotarget.13975

Figure Lengend Snippet: ( A ) Meprin α expression was analyzed by qRT-PCR in HuH7 cells following transfection of M1 or M2 meprin a siRNAs ( p = 0.0002, n =3). ( B ) Same experiment in Hep3B cells ( p = 0.0002, n =3). ( C ) Western blot of HuH7 extracts showing decreased expression of meprin α following transfection with a control siRNA (C) or with the M1 or M2 siRNAs. ( D ) Overexpression of meprin α with a V5 tag in HuH7 or Hep3B cells. Stable cell lines were established as described in Methods. ( E ) Immunofluorescence for Meprin (left) or the V5 tag (right). ( F ) Meprin α proteolytic activity in the conditioned medium is expectedly increased in HuH7 cells overexpressing meprin-V5.

Article Snippet: Quantitative RT-PCR was performed using predesigned TaqMan probes (Hs00194410_m1, Hs00195535_m1, and -Hs00272632_m1 for MEP1A, MEP1B, and RUVBL2 (Reptin), respectively) and the ABI BioMark HD reader (Fluidigm).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Over Expression, Stable Transfection, Immunofluorescence, Activity Assay

Journal: Oncotarget

Article Title: Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin

doi: 10.18632/oncotarget.13975

Figure Lengend Snippet: ( A ) HuH7 cell proliferation was assessed by direct cell counting in cells transfected with a control siRNA, or siRNAs targeting Reptin or meprin α (M1 and M2). Reptin silencing significantly decreased proliferation. The M2 siRNA had no significant effect whereas the M1 siRNA has a slight but significant effect ( p = 0.05, n = 3). ( B ) MTS assay in HuH7 cells showing no significant effect of the two meprin siRNAs on proliferation. ( C ) Cell counting experiment in Hep3B cells showing that Reptin, but not meprin silencing, reduces proliferation ( n = 3). ( D ) Growth of HuH7 (left) or Hep3B cells (right) overexpressing meprin-V5 assessed with a MTS assay ( n = 3). ( E ) Growth of HuH7 (left) or Hep3B cells (right) treated with 20 nM recombinant meprin α assessed with a MTS assay ( n = 3).

Article Snippet: Quantitative RT-PCR was performed using predesigned TaqMan probes (Hs00194410_m1, Hs00195535_m1, and -Hs00272632_m1 for MEP1A, MEP1B, and RUVBL2 (Reptin), respectively) and the ABI BioMark HD reader (Fluidigm).

Techniques: Cell Counting, Transfection, Control, MTS Assay, Recombinant

Journal: JCI Insight

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigens

doi: 10.1172/jci.insight.172449

Figure Lengend Snippet: ( A ) Proteins that were immunoprecipitated by recombinant antibody 19-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. The proteins, RUVBL1 and RUVBL2, were identified by mass spectrometry. ( B ) MiaPaca2 cells were costained with 19-3 and an antibody specific for RUVBL2. ( C ) The binding of incremental concentrations of 19-3 to RUVBL2 was measured by ELISA. ( D ) Western blot analysis of recombinant antibody 15-7 detection of cytoplasmic proteins (Cyto) and mitochondria enriched proteins (Mito) is shown. ( E ) Immunofluorescence staining of MiaPaca2 cells by 15-7 and an antibody specific for HSPD1 is shown. ( F ) The binding of incremental concentrations of 15-7 to HSPD1 was measured by ELISA. ( G ) The serum IgG titers of normal individuals ( n = 61) and PDAC patients ( n = 59) to F-actin (sera diluted 1:100), to RUVBL2 (sera diluted 1:1,000), and to HSPD1 (sera diluted 1:1,000), respectively, were measured by ELISA. Background ELISA signal is indicated by dashed line. Individual data and mean are shown. The nonparametric t test was used for comparison. Each experiment was repeated at least twice. Scale bars in B and E are 50 μm.

Article Snippet: Recombinant proteins RUVBL2 (Creative Biomart, RUVBL2-30950), RUVBL1 (Novus, NBP1-50845), HSPD1 (Origene, TP760396), or BSA control were coated in PBS with 0.25 μg per well in a 96-well ELISA plate (Corning) overnight in 4°C.

Techniques: Immunoprecipitation, Recombinant, SDS Page, Silver Staining, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Comparison