Structured Review

Santa Cruz Biotechnology anti rabbit igg
EBV infection of AGS cells suppresses GKN1 and GKN2 transcription. (A) Western blot analysis of <t>EBNA1</t> protein expression in AGS-EBV cells compared with EBV-negative AGS cells was performed using antibody for EBNA1 (top) or Actin (bottom). (B) DNA copy number was assayed by real-time PCR of EBV Ori-Lyt DNA relative to the level of cellular GAPDH in AGS, AGS-EBV, and EBV-LCL cells. (C) ChIP and real-time PCR analysis of EBNA1 (red bars) or control <t>IgG</t> (blue bars) for DNA binding at the EBV sites including DS and Ori-Lyt, or cellular sites including GKN1/2 and GAPDH in AGS-EBV cells. (D) RT-PCR was performed to analyze the mRNA level of GKN1 or GKN2 in AGS (blue bars) or AGS-EBV cells (red bars). ** indicates p
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1) Product Images from "EBNA1 binding and epigenetic regulation of gastrokine tumor suppressor genes in gastric carcinoma cells"

Article Title: EBNA1 binding and epigenetic regulation of gastrokine tumor suppressor genes in gastric carcinoma cells

Journal: Virology Journal

doi: 10.1186/1743-422X-11-12

EBV infection of AGS cells suppresses GKN1 and GKN2 transcription. (A) Western blot analysis of EBNA1 protein expression in AGS-EBV cells compared with EBV-negative AGS cells was performed using antibody for EBNA1 (top) or Actin (bottom). (B) DNA copy number was assayed by real-time PCR of EBV Ori-Lyt DNA relative to the level of cellular GAPDH in AGS, AGS-EBV, and EBV-LCL cells. (C) ChIP and real-time PCR analysis of EBNA1 (red bars) or control IgG (blue bars) for DNA binding at the EBV sites including DS and Ori-Lyt, or cellular sites including GKN1/2 and GAPDH in AGS-EBV cells. (D) RT-PCR was performed to analyze the mRNA level of GKN1 or GKN2 in AGS (blue bars) or AGS-EBV cells (red bars). ** indicates p
Figure Legend Snippet: EBV infection of AGS cells suppresses GKN1 and GKN2 transcription. (A) Western blot analysis of EBNA1 protein expression in AGS-EBV cells compared with EBV-negative AGS cells was performed using antibody for EBNA1 (top) or Actin (bottom). (B) DNA copy number was assayed by real-time PCR of EBV Ori-Lyt DNA relative to the level of cellular GAPDH in AGS, AGS-EBV, and EBV-LCL cells. (C) ChIP and real-time PCR analysis of EBNA1 (red bars) or control IgG (blue bars) for DNA binding at the EBV sites including DS and Ori-Lyt, or cellular sites including GKN1/2 and GAPDH in AGS-EBV cells. (D) RT-PCR was performed to analyze the mRNA level of GKN1 or GKN2 in AGS (blue bars) or AGS-EBV cells (red bars). ** indicates p

Techniques Used: Infection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Reverse Transcription Polymerase Chain Reaction

EBNA1 binds at the GKN1 and GKN2 promoter locus. (A) The UCSC genome browser was used to map EBNA1 binding peak generated from Raji and C666-1 ChIP-seq at the GKN1 and GKN2 gene loci. RefSeq annotated transcripts are indicated below the ChIP-Seq peak. (B-C) Realtime-PCR validation of ChIP-seq data for EBNA1 binding site at the GKN1/2 shared promoter region: EBNA1 (red bars) or control IgG (blue bars) were assayed by ChIP in Raji (B) or C666-1 cells (C) for DNA binding at the GKN1/2 site, PITPNB promoter, GAPDH, or EBV Ori-Lyt. (D) EMSA analysis of 32 P labeled probes containing Control, GKN1/2 site, or EBV FR. Varying amount of EBNA1 DBD protein was used in binding reaction (0, 100, 300, 900 ng). Arrowheads represent EBNA1-specific bound complexes or free probe as indicated. The probe sequence of GKN1/2 site and FR is indicated with sequence homolog (red letters) between GKN1/2 and FR. The negative control (Ctrl) sequence is also indicated. Error bars indicate standard deviation from the mean (sdm) for n = 3.
Figure Legend Snippet: EBNA1 binds at the GKN1 and GKN2 promoter locus. (A) The UCSC genome browser was used to map EBNA1 binding peak generated from Raji and C666-1 ChIP-seq at the GKN1 and GKN2 gene loci. RefSeq annotated transcripts are indicated below the ChIP-Seq peak. (B-C) Realtime-PCR validation of ChIP-seq data for EBNA1 binding site at the GKN1/2 shared promoter region: EBNA1 (red bars) or control IgG (blue bars) were assayed by ChIP in Raji (B) or C666-1 cells (C) for DNA binding at the GKN1/2 site, PITPNB promoter, GAPDH, or EBV Ori-Lyt. (D) EMSA analysis of 32 P labeled probes containing Control, GKN1/2 site, or EBV FR. Varying amount of EBNA1 DBD protein was used in binding reaction (0, 100, 300, 900 ng). Arrowheads represent EBNA1-specific bound complexes or free probe as indicated. The probe sequence of GKN1/2 site and FR is indicated with sequence homolog (red letters) between GKN1/2 and FR. The negative control (Ctrl) sequence is also indicated. Error bars indicate standard deviation from the mean (sdm) for n = 3.

Techniques Used: Binding Assay, Generated, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Labeling, Sequencing, Negative Control, Standard Deviation

2) Product Images from "Tyrosylprotein Sulfotransferase-1 and Tyrosine Sulfation of Chemokine Receptor 4 Are Induced by Epstein-Barr Virus Encoded Latent Membrane Protein 1 and Associated with the Metastatic Potential of Human Nasopharyngeal Carcinoma"

Article Title: Tyrosylprotein Sulfotransferase-1 and Tyrosine Sulfation of Chemokine Receptor 4 Are Induced by Epstein-Barr Virus Encoded Latent Membrane Protein 1 and Associated with the Metastatic Potential of Human Nasopharyngeal Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056114

LMP1 induces the expression of TPST-1 by activating EGFR. (A) LMP1 and TPST-1 expression was assessed in HNE2-PSG5 and HNE2-LMP1 whole cell lysates by Western blot. β-Actin was used as a control to verify equal protein loading. (B) EGFRsiRNA decreases expression of TPST-1 in Tet-on-LMP1 HNE2 cells. Tet-on-LMP1 HNE2 cells were stimulated with the indicated doses of Dox for 24 h followed by incubation with 50 pM EGFRsiRNA for an additional 24 h. EGFR and TPST-1 expression was measured in whole cell lysates by Western blot. β-Actin was used as a control to verify equal protein loading. (C) Expression level of each protein was estimated by densitometry and presented as a ratio to the loading control β-actin. The data are shown as means ± S.D. of three independent experiments performed in triplicate. (D) S-CXCR4 expression was assessed in 6–10B and 5–8F cells by (D) ChIP-PCR analysis of EGFR binding site on the promoter of TPST-1 in HNE2-PSG5 and HNE2-LMP1 cells. The cross-linked chromatin that was precipitated with specific antibodies as indicated. The input fraction represents the positive control. Negative controls include a sample with no chromatin, a sample with no antibody, and a sample with a nonspecific antibody (IgG). The precipitated DNA was analyzed by PCR using primers that amplified a 94-bp region, which included the EGFR site in HNE2-PSG5 and HNE2-LMP1 cells. (E) LMP1 promotes the binding of the EGFR and the TPST-1 promoter. ChIP-Q-PCR analysis shows that LMP1 promotes EGFR binding to the TPST-1 promoter. Anti-EGFR was used to identify the EGFR binding sites on the TPST-1 promoter in HNE2-PSG5 and HNE2-LMP1cells. The binding activity of each protein is given as percentage of total input. (F) LMP1 augments TPST-1 promoter activity through the EGFR. The constructs carry either wild-type sequences or mutations (depicted by crosses) in the EGFR sites are shown. Transient transfection and luciferase reporter assays were performed as described in “ Materials and methods ” to compare the transcriptional activation of the TPST-1 promoter in nasopharyngeal carcinoma cells. The relative luciferase activity is normalized to the value of Renilla luciferase activity. Results are expressed as fold induction of the activity of vector-transfected HNE2-PSG5 cells, which was assigned a value of 1. The data are shown as means ± S.D. of the 3 independent experiments performed in triplicate.
Figure Legend Snippet: LMP1 induces the expression of TPST-1 by activating EGFR. (A) LMP1 and TPST-1 expression was assessed in HNE2-PSG5 and HNE2-LMP1 whole cell lysates by Western blot. β-Actin was used as a control to verify equal protein loading. (B) EGFRsiRNA decreases expression of TPST-1 in Tet-on-LMP1 HNE2 cells. Tet-on-LMP1 HNE2 cells were stimulated with the indicated doses of Dox for 24 h followed by incubation with 50 pM EGFRsiRNA for an additional 24 h. EGFR and TPST-1 expression was measured in whole cell lysates by Western blot. β-Actin was used as a control to verify equal protein loading. (C) Expression level of each protein was estimated by densitometry and presented as a ratio to the loading control β-actin. The data are shown as means ± S.D. of three independent experiments performed in triplicate. (D) S-CXCR4 expression was assessed in 6–10B and 5–8F cells by (D) ChIP-PCR analysis of EGFR binding site on the promoter of TPST-1 in HNE2-PSG5 and HNE2-LMP1 cells. The cross-linked chromatin that was precipitated with specific antibodies as indicated. The input fraction represents the positive control. Negative controls include a sample with no chromatin, a sample with no antibody, and a sample with a nonspecific antibody (IgG). The precipitated DNA was analyzed by PCR using primers that amplified a 94-bp region, which included the EGFR site in HNE2-PSG5 and HNE2-LMP1 cells. (E) LMP1 promotes the binding of the EGFR and the TPST-1 promoter. ChIP-Q-PCR analysis shows that LMP1 promotes EGFR binding to the TPST-1 promoter. Anti-EGFR was used to identify the EGFR binding sites on the TPST-1 promoter in HNE2-PSG5 and HNE2-LMP1cells. The binding activity of each protein is given as percentage of total input. (F) LMP1 augments TPST-1 promoter activity through the EGFR. The constructs carry either wild-type sequences or mutations (depicted by crosses) in the EGFR sites are shown. Transient transfection and luciferase reporter assays were performed as described in “ Materials and methods ” to compare the transcriptional activation of the TPST-1 promoter in nasopharyngeal carcinoma cells. The relative luciferase activity is normalized to the value of Renilla luciferase activity. Results are expressed as fold induction of the activity of vector-transfected HNE2-PSG5 cells, which was assigned a value of 1. The data are shown as means ± S.D. of the 3 independent experiments performed in triplicate.

Techniques Used: Expressing, Western Blot, Incubation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Positive Control, Amplification, Activity Assay, Construct, Transfection, Luciferase, Activation Assay, Plasmid Preparation

3) Product Images from "FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function"

Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw498

FOXA1 and TET1 proteins physically interact. ( A ) Immunoprecipitation of ectopic FOXA1 pulled down TET1 protein. The 293T cells were transfected with Flag-TET1, either alone or together with FOXA1, for 48 h and then subjected to immunoprecipitation using an FOXA1 antibody. Whole cell (Input) and IP-enriched lysates were then analyzed by western blotting using anti-Flag (TET1) and anti-FOXA1 antibodies. ( B ) Ectopic TET1 immunoprecipitation pulled down FOXA1 protein. The 293T cells were co-transfected with FOXA1 and SFB-tagged empty vector (EV) or TET1 for 48 h before immunoprecipitation using S beads, which will pull down SFB-EV or SFB-TET1. The input and IP-enriched cell lysates were then subjected to western blotting using anti-FOXA1 and anti-Flag (for SFB-EV or SFB-TET1) antibodies. ( C ) Endogenous FOXA1 and TET1 proteins interact in LNCaP cells. LNCaP cells were subjected to immunoprecipitation using anti-FOXA1, anti-TET1 and IgG control, followed by western blotting of FOXA1 and TET1 proteins. ( D ) TET1 CXXC domain interacts with the FOXA1 protein. 293T cells were co-transfected with SFB-FOXA1 along with various Myc-tagged TET1 domain constructs. The expression of TET1 domains in whole cell lysate (input) was confirmed by western blotting using anti-Myc. Cell lysates were then subjected to S pull down (of FOXA1) and subsequently western blot analysis using anti-FOXA1 and anti-Myc antibodies. ( E ) FOXA1 FH (Forkhead-containing) domain interacts with TET1 CXXC domain. 293T cells were co-transfected with SFB-CXXC along with various Flag-tagged FOXA1 domain constructs and subjected to S pull down (of TET1-CXXC) followed by western blotting using an anti-Flag antibody. ( F ) In vitro interaction assay was conducted using purified proteins of TET1 CXXC domain and FOXA1 Forkhead domain. CXXC domain was tagged with GST and further subdivided into fragments A and B (the ‘C-X-X-C’ motif was located in residues 590–609 in fragment A), and FH domain was tagged with Myc. Arrows point to expression of proteins according to their expected size.
Figure Legend Snippet: FOXA1 and TET1 proteins physically interact. ( A ) Immunoprecipitation of ectopic FOXA1 pulled down TET1 protein. The 293T cells were transfected with Flag-TET1, either alone or together with FOXA1, for 48 h and then subjected to immunoprecipitation using an FOXA1 antibody. Whole cell (Input) and IP-enriched lysates were then analyzed by western blotting using anti-Flag (TET1) and anti-FOXA1 antibodies. ( B ) Ectopic TET1 immunoprecipitation pulled down FOXA1 protein. The 293T cells were co-transfected with FOXA1 and SFB-tagged empty vector (EV) or TET1 for 48 h before immunoprecipitation using S beads, which will pull down SFB-EV or SFB-TET1. The input and IP-enriched cell lysates were then subjected to western blotting using anti-FOXA1 and anti-Flag (for SFB-EV or SFB-TET1) antibodies. ( C ) Endogenous FOXA1 and TET1 proteins interact in LNCaP cells. LNCaP cells were subjected to immunoprecipitation using anti-FOXA1, anti-TET1 and IgG control, followed by western blotting of FOXA1 and TET1 proteins. ( D ) TET1 CXXC domain interacts with the FOXA1 protein. 293T cells were co-transfected with SFB-FOXA1 along with various Myc-tagged TET1 domain constructs. The expression of TET1 domains in whole cell lysate (input) was confirmed by western blotting using anti-Myc. Cell lysates were then subjected to S pull down (of FOXA1) and subsequently western blot analysis using anti-FOXA1 and anti-Myc antibodies. ( E ) FOXA1 FH (Forkhead-containing) domain interacts with TET1 CXXC domain. 293T cells were co-transfected with SFB-CXXC along with various Flag-tagged FOXA1 domain constructs and subjected to S pull down (of TET1-CXXC) followed by western blotting using an anti-Flag antibody. ( F ) In vitro interaction assay was conducted using purified proteins of TET1 CXXC domain and FOXA1 Forkhead domain. CXXC domain was tagged with GST and further subdivided into fragments A and B (the ‘C-X-X-C’ motif was located in residues 590–609 in fragment A), and FH domain was tagged with Myc. Arrows point to expression of proteins according to their expected size.

Techniques Used: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, Construct, Expressing, In Vitro, Purification

TET1 is a direct transcriptional target of FOXA1. ( A ) ChIP-seq showing FOXA1 binding events at TET1 promoter and enhancer. FOXA1 ChIP-seq was conducted in LNCaP cells and FOXA1 binding events were identified by HOMER and visualized in UCSC Genome Browser. FKHD motifs (indicated by red box) near FXBS were determined by JASPAR. DNA fragments containing FXBS at the TET1 promoter (pTET1) and enhancer (eTET1) were each cloned into pGL4 luciferase reporter construct with wild-type (WT) or mutated (mut) FKHD motif (mutated nt shown in red at the bottom panel). ( B ) ChIP-PCR validation of FOXA1 binding to TET1 enhancer and promoter in LNCaP cells. ChIP was performed using anti-FOXA1 and anti-IgG antibodies in LNCaP cells. ChIP-qPCR was performed using primers flanking the FOXA1 binding peaks at the TET1 enhancer (eTET1) and promoter (pTET). PSA is used as a positive control while KIAA0066 a negative control. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. ( C ) FOXA1 occupancy at TET1 promoter and enhancer was decreased by FOXA1 knockdown. ChIP-qPCR using anti-FOXA1 antibody was carried out in control and FOXA1-depleted LNCaP cells. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. * P
Figure Legend Snippet: TET1 is a direct transcriptional target of FOXA1. ( A ) ChIP-seq showing FOXA1 binding events at TET1 promoter and enhancer. FOXA1 ChIP-seq was conducted in LNCaP cells and FOXA1 binding events were identified by HOMER and visualized in UCSC Genome Browser. FKHD motifs (indicated by red box) near FXBS were determined by JASPAR. DNA fragments containing FXBS at the TET1 promoter (pTET1) and enhancer (eTET1) were each cloned into pGL4 luciferase reporter construct with wild-type (WT) or mutated (mut) FKHD motif (mutated nt shown in red at the bottom panel). ( B ) ChIP-PCR validation of FOXA1 binding to TET1 enhancer and promoter in LNCaP cells. ChIP was performed using anti-FOXA1 and anti-IgG antibodies in LNCaP cells. ChIP-qPCR was performed using primers flanking the FOXA1 binding peaks at the TET1 enhancer (eTET1) and promoter (pTET). PSA is used as a positive control while KIAA0066 a negative control. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. ( C ) FOXA1 occupancy at TET1 promoter and enhancer was decreased by FOXA1 knockdown. ChIP-qPCR using anti-FOXA1 antibody was carried out in control and FOXA1-depleted LNCaP cells. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Clone Assay, Luciferase, Construct, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

4) Product Images from "The Transcriptional Co-Regulator HCF-1 Is Required for INS-1 ?-cell Glucose-Stimulated Insulin Secretion"

Article Title: The Transcriptional Co-Regulator HCF-1 Is Required for INS-1 ?-cell Glucose-Stimulated Insulin Secretion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0078841

HCF-1 and E2F1 occupy the Pdx1 promoter. INS-1 cells treated with HCF-1 siRNA or control siRNA were subjected to chromatin immunoprecipitation assays using antibodies directed against (A) HCF-1, (B) E2F1 or isotype-matched rabbit IgG. Immunoprecipitated DNA was quantified using qPCR and calculated as percent of input DNA. Results shown are pooled from three independent experiments and represent the mean +/− SEM. HCF-1 and E2F1 enrichment at the Pdx1 and cyclin A2 promoters was significantly greater than at the negative control region (chrIII) (*denotes a p-value
Figure Legend Snippet: HCF-1 and E2F1 occupy the Pdx1 promoter. INS-1 cells treated with HCF-1 siRNA or control siRNA were subjected to chromatin immunoprecipitation assays using antibodies directed against (A) HCF-1, (B) E2F1 or isotype-matched rabbit IgG. Immunoprecipitated DNA was quantified using qPCR and calculated as percent of input DNA. Results shown are pooled from three independent experiments and represent the mean +/− SEM. HCF-1 and E2F1 enrichment at the Pdx1 and cyclin A2 promoters was significantly greater than at the negative control region (chrIII) (*denotes a p-value

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

HCF-1 is required for glucose-stimulated insulin secretion. (A) Immunohistochemistry showing HCF-1 expression in mouse pancreas. Pancreas from 6-week-old male C57BL/6 mice was stained with HCF-1 (left panel) or IgG-control (right panel). HCF-1 is expressed in the nuclei of cells from the exocrine pancreas and the islet of Langerhans. (B) Western blot analysis of HCF-1 protein levels from INS-1 cells transfected twice with control or four different HCF-1 siRNAs. (C) Western blot analysis of HCF-1 levels from cells induced to express shLuc or shHCF-1 shRNA with doxycycline treatment. (D) Glucose-stimulated insulin secretion analysis of cells treated with HCF-1 siRNA. Insulin secretion determined by insulin ELISA and normalized to total cellular protein. Results shown are pooled from three independent experiments, and represent mean +/− SEM. *denotes a p-value
Figure Legend Snippet: HCF-1 is required for glucose-stimulated insulin secretion. (A) Immunohistochemistry showing HCF-1 expression in mouse pancreas. Pancreas from 6-week-old male C57BL/6 mice was stained with HCF-1 (left panel) or IgG-control (right panel). HCF-1 is expressed in the nuclei of cells from the exocrine pancreas and the islet of Langerhans. (B) Western blot analysis of HCF-1 protein levels from INS-1 cells transfected twice with control or four different HCF-1 siRNAs. (C) Western blot analysis of HCF-1 levels from cells induced to express shLuc or shHCF-1 shRNA with doxycycline treatment. (D) Glucose-stimulated insulin secretion analysis of cells treated with HCF-1 siRNA. Insulin secretion determined by insulin ELISA and normalized to total cellular protein. Results shown are pooled from three independent experiments, and represent mean +/− SEM. *denotes a p-value

Techniques Used: Immunohistochemistry, Expressing, Mouse Assay, Staining, Western Blot, Transfection, shRNA, Enzyme-linked Immunosorbent Assay

5) Product Images from "Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *"

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.677898

Analysis of the NCX1, PP1c, and PLM macromolecular complex in HEK293 cells. A, HEK293 cells were co-transfected with PLM and NCX1(FL)-GFP. The lysates were subjected to immunoprecipitation ( IP ) using anti-NCX1. Immunoprecipitates ( right panels ) and lysate ( input ; positive control for the immunoblot, left panel ) were immunoblotted with NCX1, PP1c, and PLM antibodies. As a negative control NCX1 antibody pre-incubated with a specific anti-NCX1 blocking peptide or non-relevant rabbit IgG was used. B, epitope mapping was performed by overlaying an array of immobilized overlapping 20-mer PLM peptides with anti-pSer-68-PLM ( right panel ) ( n ). Immunodetection without anti-pSer-68-PLM was used as a negative control ( left panel ). FLAG-His 6 -PP1c(1–330) and PLM were expressed with and without either NCX(FL)-GFP ( C ) or GFP-NCX1(F407P) ( D ), representing the proline substitution KIPF, or GFP-NCX1(Δ399–424), representing KIFF deletion, in HEK293 followed by immunoblotting with anti-NCX1, FLAG, pSer-68-PLM, and total PLM. Non-transfected HEK293 cells were used as control in C. Bar graph shows relative pSer-68-PLM/total PLM level quantified by densitometry analysis. Differences were tested using unpaired t test (**, p
Figure Legend Snippet: Analysis of the NCX1, PP1c, and PLM macromolecular complex in HEK293 cells. A, HEK293 cells were co-transfected with PLM and NCX1(FL)-GFP. The lysates were subjected to immunoprecipitation ( IP ) using anti-NCX1. Immunoprecipitates ( right panels ) and lysate ( input ; positive control for the immunoblot, left panel ) were immunoblotted with NCX1, PP1c, and PLM antibodies. As a negative control NCX1 antibody pre-incubated with a specific anti-NCX1 blocking peptide or non-relevant rabbit IgG was used. B, epitope mapping was performed by overlaying an array of immobilized overlapping 20-mer PLM peptides with anti-pSer-68-PLM ( right panel ) ( n ). Immunodetection without anti-pSer-68-PLM was used as a negative control ( left panel ). FLAG-His 6 -PP1c(1–330) and PLM were expressed with and without either NCX(FL)-GFP ( C ) or GFP-NCX1(F407P) ( D ), representing the proline substitution KIPF, or GFP-NCX1(Δ399–424), representing KIFF deletion, in HEK293 followed by immunoblotting with anti-NCX1, FLAG, pSer-68-PLM, and total PLM. Non-transfected HEK293 cells were used as control in C. Bar graph shows relative pSer-68-PLM/total PLM level quantified by densitometry analysis. Differences were tested using unpaired t test (**, p

Techniques Used: Transfection, Immunoprecipitation, Positive Control, Negative Control, Incubation, Blocking Assay, Immunodetection

6) Product Images from "Mutations in the Cholesterol Transporter Gene ABCA5 Are Associated with Excessive Hair Overgrowth"

Article Title: Mutations in the Cholesterol Transporter Gene ABCA5 Are Associated with Excessive Hair Overgrowth

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004333

ABCA5 protein levels are significantly reduced in CGHT patient keratinocytes and hair follicles. (A) Immunofluorescence staining on cultured keratinocytes revealed a dramatic decrease in ABCA5 localization in CGHT keratinocytes compared to control (B) Immunoblotting on protein extracted from control and patient keratinocytes demonstrated loss of a 215 kDa band, which corresponds to the glycosylated form of the full-length ABCA5 protein, as well as a 187 kDa band, which is the unmodified protein in the patient compared to control. (C) Loss of ABCA5 localization to the outer root sheath (ORS) and inner root sheath (IRS) within patient hair follicles by immunofluorescence staining. Affected hair follicles in anagen and catagen were obtained from forearm skin biopsies, and control catagen hairs were obtained from forearm skin and anagen hairs from the occipital scalp. The anti-rabbit IgG primary antibody control produced no signal. HS = hair shaft; DP = dermal papilla; DS = dermal sheath.
Figure Legend Snippet: ABCA5 protein levels are significantly reduced in CGHT patient keratinocytes and hair follicles. (A) Immunofluorescence staining on cultured keratinocytes revealed a dramatic decrease in ABCA5 localization in CGHT keratinocytes compared to control (B) Immunoblotting on protein extracted from control and patient keratinocytes demonstrated loss of a 215 kDa band, which corresponds to the glycosylated form of the full-length ABCA5 protein, as well as a 187 kDa band, which is the unmodified protein in the patient compared to control. (C) Loss of ABCA5 localization to the outer root sheath (ORS) and inner root sheath (IRS) within patient hair follicles by immunofluorescence staining. Affected hair follicles in anagen and catagen were obtained from forearm skin biopsies, and control catagen hairs were obtained from forearm skin and anagen hairs from the occipital scalp. The anti-rabbit IgG primary antibody control produced no signal. HS = hair shaft; DP = dermal papilla; DS = dermal sheath.

Techniques Used: Immunofluorescence, Staining, Cell Culture, Produced

The ABCA5 c.4320+1G > C mutation leads to aberrant splicing and nonsense mediated decay, and ABCA5 is abundantly expressed in the skin and hair follicle. (A) Depiction of normal splicing between exons 31 and 32 (black lines) and aberrant skipping of exon 32 as a result of the c.4320+1G > C mutation (red lines), where the adjoining of exon 31 to 33 leads to premature termination during translation. The out-of-frame exon 33 sequence (red box) is indicated by the hatched lines, followed by a stop codon. Sequencing of the mutant transcript (MT) confirmed the aberrant splicing event, where the amino acid sequence is listed above and the stop codon is indicated by an asterisk. RT-PCR on RNA from whole skin followed by gel electrophoresis of the exon 31–33 amplicon demonstrates complete loss of the wild-type (WT) transcript in the patient (P), and very low levels of the mutant (MT) transcript in the carrier (C). (B) qRT-PCR of ABCA5 transcript levels in the carrier and patient revealed a significant reduction of levels in patient whole skin, keratinocytes and fibroblasts. A Student unpaired t test was performed with a cutoff P value of 0.05 for statistical significance. (C) ABCA5 is expressed at strong levels throughout the outer root sheath (ORS) and dermal sheath (arrow) of the human hair follicle by in situ hybridization using an antisense probe, whereas the sense probe produced minimal background signal. (D) ABCA5 localizes to the perifollicular dermis, dermal sheath, as well as the hair follicle ORS and inner root sheath (IRS) by immunohistochemistry on paraffin-embedded sections. (E) The anti-rabbit IgG primary antibody control produced no signal. (F) Immunohistochemistry of ABCA5 on hair follicle end bulbs revealed strong expression in the dermal sheath and perifolliclar dermis but no expression was observed in the dermal papilla. (G) ABCA5 is endogenously expressed in plucked scalp hair follicles, microdissected ORS, as well as perifollicular dermis (including dermal sheath), determined by RT-PCR. Expression was normalized to GAPDH levels. M = marker.
Figure Legend Snippet: The ABCA5 c.4320+1G > C mutation leads to aberrant splicing and nonsense mediated decay, and ABCA5 is abundantly expressed in the skin and hair follicle. (A) Depiction of normal splicing between exons 31 and 32 (black lines) and aberrant skipping of exon 32 as a result of the c.4320+1G > C mutation (red lines), where the adjoining of exon 31 to 33 leads to premature termination during translation. The out-of-frame exon 33 sequence (red box) is indicated by the hatched lines, followed by a stop codon. Sequencing of the mutant transcript (MT) confirmed the aberrant splicing event, where the amino acid sequence is listed above and the stop codon is indicated by an asterisk. RT-PCR on RNA from whole skin followed by gel electrophoresis of the exon 31–33 amplicon demonstrates complete loss of the wild-type (WT) transcript in the patient (P), and very low levels of the mutant (MT) transcript in the carrier (C). (B) qRT-PCR of ABCA5 transcript levels in the carrier and patient revealed a significant reduction of levels in patient whole skin, keratinocytes and fibroblasts. A Student unpaired t test was performed with a cutoff P value of 0.05 for statistical significance. (C) ABCA5 is expressed at strong levels throughout the outer root sheath (ORS) and dermal sheath (arrow) of the human hair follicle by in situ hybridization using an antisense probe, whereas the sense probe produced minimal background signal. (D) ABCA5 localizes to the perifollicular dermis, dermal sheath, as well as the hair follicle ORS and inner root sheath (IRS) by immunohistochemistry on paraffin-embedded sections. (E) The anti-rabbit IgG primary antibody control produced no signal. (F) Immunohistochemistry of ABCA5 on hair follicle end bulbs revealed strong expression in the dermal sheath and perifolliclar dermis but no expression was observed in the dermal papilla. (G) ABCA5 is endogenously expressed in plucked scalp hair follicles, microdissected ORS, as well as perifollicular dermis (including dermal sheath), determined by RT-PCR. Expression was normalized to GAPDH levels. M = marker.

Techniques Used: Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification, Quantitative RT-PCR, In Situ Hybridization, Produced, Immunohistochemistry, Expressing, Marker

7) Product Images from "The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration"

Article Title: The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration

Journal: Cancers

doi: 10.3390/cancers11121991

α9-nAChR mediates nicotine-induced PD-L1 expression and regulates melanoma cell proliferation. ( A ) Nicotine increased STAT3, AKT, and ERK phosphorylation in a time-dependent manner in the A375, A2058, and MDA-MB-435 cells, as assessed by western blotting. ( B ) Nicotine induced the phosphorylation of STAT3, AKT, and ERK and upregulated the protein levels of α9-nAChR, PD-L1, Snail-1 and Twist-1 in a dose-dependent manner. The results were determined by western blotting. ( C – J ) A2058 cells were pretreated with the inhibitor LY294002 (10 μm or 25 μm), PD98059 (10 μm or 25 μm), Stattic (2.5 μm or 5 μm) or NSC74859 (10 μm or 25 μm) for 3-24 h and subsequently treated with or without nicotine (1 μm) for additional 24-48 h. Western blot analysis was used to detect protein levels. Cell proliferation was assessed by an MTT assay. ( K , L ) Wild-type, scrambled siRNA, and α9-nAChR-si-expressing A2058 cells were treated with or without 1 μm nicotine. Western blot analysis was used to assess protein levels. Cell growth was assessed by an MTT assay. ( M , N ) After A2058 cells were exposed to 0.1 μm or 1 μm nicotine for 24 h, a ChIP assay was performed using an anti-STAT3 antibody. The protein-chromatin immunoprecipitates were subjected to RT-PCR and qPCR. In RT-PCR, a rabbit IgG antibody was used as a negative control, and total genomic DNA was used as a positive control. In qPCR, relative enrichments of PD-L1 promoter region were calculated after normalization to GAPDH. Each of the immunoprecipitations was replicated three times, and each sample was quantified at least in triplicate. The data are presented as the mean ± SD of three independent experiments. ns, not significant; * p
Figure Legend Snippet: α9-nAChR mediates nicotine-induced PD-L1 expression and regulates melanoma cell proliferation. ( A ) Nicotine increased STAT3, AKT, and ERK phosphorylation in a time-dependent manner in the A375, A2058, and MDA-MB-435 cells, as assessed by western blotting. ( B ) Nicotine induced the phosphorylation of STAT3, AKT, and ERK and upregulated the protein levels of α9-nAChR, PD-L1, Snail-1 and Twist-1 in a dose-dependent manner. The results were determined by western blotting. ( C – J ) A2058 cells were pretreated with the inhibitor LY294002 (10 μm or 25 μm), PD98059 (10 μm or 25 μm), Stattic (2.5 μm or 5 μm) or NSC74859 (10 μm or 25 μm) for 3-24 h and subsequently treated with or without nicotine (1 μm) for additional 24-48 h. Western blot analysis was used to detect protein levels. Cell proliferation was assessed by an MTT assay. ( K , L ) Wild-type, scrambled siRNA, and α9-nAChR-si-expressing A2058 cells were treated with or without 1 μm nicotine. Western blot analysis was used to assess protein levels. Cell growth was assessed by an MTT assay. ( M , N ) After A2058 cells were exposed to 0.1 μm or 1 μm nicotine for 24 h, a ChIP assay was performed using an anti-STAT3 antibody. The protein-chromatin immunoprecipitates were subjected to RT-PCR and qPCR. In RT-PCR, a rabbit IgG antibody was used as a negative control, and total genomic DNA was used as a positive control. In qPCR, relative enrichments of PD-L1 promoter region were calculated after normalization to GAPDH. Each of the immunoprecipitations was replicated three times, and each sample was quantified at least in triplicate. The data are presented as the mean ± SD of three independent experiments. ns, not significant; * p

Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, MTT Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

8) Product Images from "A Novel Zinc Finger Protein Zfp277 Mediates Transcriptional Repression of the Ink4a/Arf Locus through Polycomb Repressive Complex 1"

Article Title: A Novel Zinc Finger Protein Zfp277 Mediates Transcriptional Repression of the Ink4a/Arf Locus through Polycomb Repressive Complex 1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012373

Zfp277 interacts physically with Bmi1. A. Zfp277 interacts with Bmi1 in vivo . HA-Zfp277 and Flag-tagged Bmi1, Ring1B, Ezh2, and Myc-tagged Mel18 were cotransfected into human 293T cells and the immunoprecipitates were subjected to a Western blot analysis using the antibodies indicated on the left. B. GST-pulldown between GST-Bmi1 and in vitro -translated Zfp277 (left panel) and GST-Zfp277 and in vitro -translated Mel18 (right panel). C. Binding of Zfp277 with PRC1 in MEFs. The whole-cell extracts from MEFs were immunoprecipitated with rabbit anti-Zfp277 serum (left panel) and anti-Bmi1 monoclonal antibody (right panel) or control IgG (control) and then subjected to Western blotting using the antibodies indicated on the left. A 2% input was loaded on the left. D. The growth curve of wild-type and Zfp277 −/− MEFs. MEFs were seeded at 1×10 5 cells/well in 6-cm plates and replated at 1×10 5 cells/well every three days. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. E. Quantitative RT-PCR analysis of p19 Arf and p16 Ink4a mRNA in MEFs at passage 2. mRNA levels were normalized to Hprt1 expression. Expression levels relative to those in the wild-type MEFs are shown as the mean ± SD for three independent experiments. F. Expression of p19 Arf and p16 Ink4a in Zfp277 −/− MEFs. The protein levels of p19 Arf and p16 Ink4a in wild-type and Zfp277 −/− MEFs at the indicated passages were determined by Western blot analyses. Tubulin was used as a loading control. G. Rescue of the growth of Zfp277 −/− MEFs by E6. Wild-type and Zfp277 −/− MEFs were transduced with either a control or an E6 retrovirus and cell growth was monitored every three days by replating at 1×10 5 cells/plate. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. Statistical significance was determined with Student's t-test; ** p
Figure Legend Snippet: Zfp277 interacts physically with Bmi1. A. Zfp277 interacts with Bmi1 in vivo . HA-Zfp277 and Flag-tagged Bmi1, Ring1B, Ezh2, and Myc-tagged Mel18 were cotransfected into human 293T cells and the immunoprecipitates were subjected to a Western blot analysis using the antibodies indicated on the left. B. GST-pulldown between GST-Bmi1 and in vitro -translated Zfp277 (left panel) and GST-Zfp277 and in vitro -translated Mel18 (right panel). C. Binding of Zfp277 with PRC1 in MEFs. The whole-cell extracts from MEFs were immunoprecipitated with rabbit anti-Zfp277 serum (left panel) and anti-Bmi1 monoclonal antibody (right panel) or control IgG (control) and then subjected to Western blotting using the antibodies indicated on the left. A 2% input was loaded on the left. D. The growth curve of wild-type and Zfp277 −/− MEFs. MEFs were seeded at 1×10 5 cells/well in 6-cm plates and replated at 1×10 5 cells/well every three days. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. E. Quantitative RT-PCR analysis of p19 Arf and p16 Ink4a mRNA in MEFs at passage 2. mRNA levels were normalized to Hprt1 expression. Expression levels relative to those in the wild-type MEFs are shown as the mean ± SD for three independent experiments. F. Expression of p19 Arf and p16 Ink4a in Zfp277 −/− MEFs. The protein levels of p19 Arf and p16 Ink4a in wild-type and Zfp277 −/− MEFs at the indicated passages were determined by Western blot analyses. Tubulin was used as a loading control. G. Rescue of the growth of Zfp277 −/− MEFs by E6. Wild-type and Zfp277 −/− MEFs were transduced with either a control or an E6 retrovirus and cell growth was monitored every three days by replating at 1×10 5 cells/plate. Cumulative cell numbers are shown as the mean ± SD for three independent triplicate experiments. Statistical significance was determined with Student's t-test; ** p

Techniques Used: In Vivo, Western Blot, In Vitro, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Expressing, Transduction

9) Product Images from "LL5? Directs the Translocation of Filamin A and SHIP2 to Sites of Phosphatidylinositol 3,4,5-Triphosphate (PtdIns(3,4,5)P3) Accumulation, and PtdIns(3,4,5)P3 Localization Is Mutually Modified by Co-recruited SHIP2 *"

Article Title: LL5? Directs the Translocation of Filamin A and SHIP2 to Sites of Phosphatidylinositol 3,4,5-Triphosphate (PtdIns(3,4,5)P3) Accumulation, and PtdIns(3,4,5)P3 Localization Is Mutually Modified by Co-recruited SHIP2 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.081901

LL5β binds to Filamin A through its CH1CH2 or repeat 24. A , distribution of Filamin A in COS-7 cells without exogenous LL5β. Scale bar = 10 μm. B , left and middle columns , following EGF application, exogenous EGFP-LL5β ( green ) and endogenous Filamin A ( red ) colocalized at the plasma membrane ( arrowheads ). Right column , endogenous Filamin A ( red ) and endogenous LL5β ( green ) were visualized in COS-7 cells that had been cultured without serum for 18 h. They accumulated near the plasma membrane at 10 min after the addition of 10% serum. Scale bar = 10 μm. C , following EGF application, exogenous EGFP-tagged ARNO/PH (EGFP-ARNO/PH) ( green ) and endogenous LL5β ( red ) colocalized at the plasma membrane ( arrowheads ). Scale bar = 10 μm. D , lysates from COS-7 cells expressing EGFP-LL5β were subjected to immunoprecipitation ( IP ) with an anti-GFP antibody or rabbit IgG (control). Endogenous Filamin A (∼280 kDa, arrow ) was co-immunoprecipitated with EGFP-LL5β. E , schematic drawings show the various truncated Filamin A mutants. They were tagged with HA at their amino termini. EGFP-LL5β was co-expressed in COS-7 cells with one of the truncated Filamin A mutants, as shown on the left , followed by immunoprecipitation with anti-GFP antibody or rabbit IgG (control) and detection with anti-HA antibody. Filamin A mutants (Filamin AΔ1-Δ3) were co-immunoprecipitated. CH1 and CH2 , actin-binding site 1 and 2, respectively. H1 and H2 , hinge 1 and 2. Numbers (1–24) indicate the FR number. F , CH1CH2, FR24, or FR23H2 (Filamin repeat 23 and hinge 2) tagged with V5 were co-expressed with EGFP-LL5β. EGFP-LL5β was co-immunoprecipitated with CH1CH2 or FR24 but not FR23H2 ( arrowhead ). G , full-length Filamin A ( arrowhead ) or Filamin AΔABD ( arrow ) tagged with HA was co-immunoprecipitated with EGFP-LL5 β. H , full-length Filamin A ( arrowhead ) or Filamin A ΔFR24 ( arrow ) tagged with HA was co-immunoprecipitated with EGFP-LL5β.
Figure Legend Snippet: LL5β binds to Filamin A through its CH1CH2 or repeat 24. A , distribution of Filamin A in COS-7 cells without exogenous LL5β. Scale bar = 10 μm. B , left and middle columns , following EGF application, exogenous EGFP-LL5β ( green ) and endogenous Filamin A ( red ) colocalized at the plasma membrane ( arrowheads ). Right column , endogenous Filamin A ( red ) and endogenous LL5β ( green ) were visualized in COS-7 cells that had been cultured without serum for 18 h. They accumulated near the plasma membrane at 10 min after the addition of 10% serum. Scale bar = 10 μm. C , following EGF application, exogenous EGFP-tagged ARNO/PH (EGFP-ARNO/PH) ( green ) and endogenous LL5β ( red ) colocalized at the plasma membrane ( arrowheads ). Scale bar = 10 μm. D , lysates from COS-7 cells expressing EGFP-LL5β were subjected to immunoprecipitation ( IP ) with an anti-GFP antibody or rabbit IgG (control). Endogenous Filamin A (∼280 kDa, arrow ) was co-immunoprecipitated with EGFP-LL5β. E , schematic drawings show the various truncated Filamin A mutants. They were tagged with HA at their amino termini. EGFP-LL5β was co-expressed in COS-7 cells with one of the truncated Filamin A mutants, as shown on the left , followed by immunoprecipitation with anti-GFP antibody or rabbit IgG (control) and detection with anti-HA antibody. Filamin A mutants (Filamin AΔ1-Δ3) were co-immunoprecipitated. CH1 and CH2 , actin-binding site 1 and 2, respectively. H1 and H2 , hinge 1 and 2. Numbers (1–24) indicate the FR number. F , CH1CH2, FR24, or FR23H2 (Filamin repeat 23 and hinge 2) tagged with V5 were co-expressed with EGFP-LL5β. EGFP-LL5β was co-immunoprecipitated with CH1CH2 or FR24 but not FR23H2 ( arrowhead ). G , full-length Filamin A ( arrowhead ) or Filamin AΔABD ( arrow ) tagged with HA was co-immunoprecipitated with EGFP-LL5 β. H , full-length Filamin A ( arrowhead ) or Filamin A ΔFR24 ( arrow ) tagged with HA was co-immunoprecipitated with EGFP-LL5β.

Techniques Used: Cell Culture, Expressing, Immunoprecipitation, Binding Assay

10) Product Images from "The interplay between the lysine demethylase KDM1A and DNA methyltransferases in cancer cells is cell cycle dependent"

Article Title: The interplay between the lysine demethylase KDM1A and DNA methyltransferases in cancer cells is cell cycle dependent

Journal: Oncotarget

doi: 10.18632/oncotarget.10624

Interaction of KDM1A with DNMT1 and DNMT3B in vitro and in cancer cells ( A ) KDM1A interacts with DNMT1 and DNMT3B in vitro . Upper panel: Schematic representation of the KDM1A protein, with its known domains highlighted. Also shown are the different sequences that were fused to GST and tested for binding to DNMT1 or DNMT3B. The results are summarized on the right (from “++” [strong interaction] to “–” [no interaction]). Middle and lower panels: The indicated GST fusions were tested in GST pull-down experiments using IVT full-length DNMT1 (IVT-DNMT1) (middle panel) or DNMT3B (IVT-DNMT3B) (lower panel) (lanes 3 to 7). Lane 2 shows the results of the control pull-down with GST protein alone. Lane 1 shows 10% of the radiolabeled IVT-DNMT1 or IVT-DNMT3B engaged in the pull-down experiment. A vertical line indicates juxtaposition of non-adjacent lanes of the same blot (the exposure time was the same). ( B ) Western blots showing that DNMT1 and DNMT3B co-immunoprecipitate with KDM1A from HCT116 whole-cell extracts (lanes 3). Anti-rabbit IgG was used in the negative control (lanes 2). Input stands for non-immunoprecipitated HCT116 extract (10% of the volume subjected to immunoprecipitation) (lanes 1). The western blot at the bottom shows the efficiency of KDM1A immunoprecipitation by the anti-KDM1A antibody. The vertical line indicates juxtaposition of non-adjacent lanes of the same blot (exposure time was the same).
Figure Legend Snippet: Interaction of KDM1A with DNMT1 and DNMT3B in vitro and in cancer cells ( A ) KDM1A interacts with DNMT1 and DNMT3B in vitro . Upper panel: Schematic representation of the KDM1A protein, with its known domains highlighted. Also shown are the different sequences that were fused to GST and tested for binding to DNMT1 or DNMT3B. The results are summarized on the right (from “++” [strong interaction] to “–” [no interaction]). Middle and lower panels: The indicated GST fusions were tested in GST pull-down experiments using IVT full-length DNMT1 (IVT-DNMT1) (middle panel) or DNMT3B (IVT-DNMT3B) (lower panel) (lanes 3 to 7). Lane 2 shows the results of the control pull-down with GST protein alone. Lane 1 shows 10% of the radiolabeled IVT-DNMT1 or IVT-DNMT3B engaged in the pull-down experiment. A vertical line indicates juxtaposition of non-adjacent lanes of the same blot (the exposure time was the same). ( B ) Western blots showing that DNMT1 and DNMT3B co-immunoprecipitate with KDM1A from HCT116 whole-cell extracts (lanes 3). Anti-rabbit IgG was used in the negative control (lanes 2). Input stands for non-immunoprecipitated HCT116 extract (10% of the volume subjected to immunoprecipitation) (lanes 1). The western blot at the bottom shows the efficiency of KDM1A immunoprecipitation by the anti-KDM1A antibody. The vertical line indicates juxtaposition of non-adjacent lanes of the same blot (exposure time was the same).

Techniques Used: In Vitro, Binding Assay, Western Blot, Negative Control, Immunoprecipitation

11) Product Images from "Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release"

Article Title: Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

Journal: PLoS ONE

doi: 10.1371/journal.pone.0094574

Ionomycin-induced Pyk2 tyrosine phosphorylation is site-specific only for Tyr-402. ( A ) PC12 cells were exposed to 1 μM ionomycin at 37°C for 90 s. The cells were fixed, and phosphotyrosine proteins were labeled by phosphorylation site-specific antibodies against Tyr-402 (pY402), Tyr-579 (pY579), Tyr-580 (pY580), or Tyr-881 (pY881) and TRITC-conjugated second antibody (red), respectively. Pyk2 expression was labeled by FITC (green). Scale bar, 5 μm. ( B ) PC12 cells were treated with or without 1 μM ionomycin at 37°C for 90 s, then lysed, and subjected to western blot using phosphorylation site-specific antibodies. Pyk2 transfected 293T cell lysate was used as a positive control. The total amount of Pyk2 was used as an internal control. ( C ) The time course of Pyk2 phosphorylation at Tyr-402 by ionomycin in PC12 cells. PC12 cells were exposed to 1 μM ionomycin at 37°C for the indicated times. The cells were labeled with pY402 site-specific polyclonal antibody, followed by a secondary layer of TRITC-conjugated second antibody to rabbit IgG (red). Scale bar, 10 μm.
Figure Legend Snippet: Ionomycin-induced Pyk2 tyrosine phosphorylation is site-specific only for Tyr-402. ( A ) PC12 cells were exposed to 1 μM ionomycin at 37°C for 90 s. The cells were fixed, and phosphotyrosine proteins were labeled by phosphorylation site-specific antibodies against Tyr-402 (pY402), Tyr-579 (pY579), Tyr-580 (pY580), or Tyr-881 (pY881) and TRITC-conjugated second antibody (red), respectively. Pyk2 expression was labeled by FITC (green). Scale bar, 5 μm. ( B ) PC12 cells were treated with or without 1 μM ionomycin at 37°C for 90 s, then lysed, and subjected to western blot using phosphorylation site-specific antibodies. Pyk2 transfected 293T cell lysate was used as a positive control. The total amount of Pyk2 was used as an internal control. ( C ) The time course of Pyk2 phosphorylation at Tyr-402 by ionomycin in PC12 cells. PC12 cells were exposed to 1 μM ionomycin at 37°C for the indicated times. The cells were labeled with pY402 site-specific polyclonal antibody, followed by a secondary layer of TRITC-conjugated second antibody to rabbit IgG (red). Scale bar, 10 μm.

Techniques Used: Labeling, Expressing, Western Blot, Transfection, Positive Control

12) Product Images from "Necdin, a Prader-Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development"

Article Title: Necdin, a Prader-Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development

Journal:

doi: 10.1093/hmg/ddn344

Necdin interacts with Msx in GT1-7 cells. ( A ) Proteins from GT1-7 cells were immunoprecipitated (IP) with either anti-necdin antibody or rabbit IgG control. Msx1 and necdin were detected by immunoblotting the immunoprecipitated proteins. Ten percent of
Figure Legend Snippet: Necdin interacts with Msx in GT1-7 cells. ( A ) Proteins from GT1-7 cells were immunoprecipitated (IP) with either anti-necdin antibody or rabbit IgG control. Msx1 and necdin were detected by immunoblotting the immunoprecipitated proteins. Ten percent of

Techniques Used: Immunoprecipitation

13) Product Images from "Expression of Membrane-bound Carbonic Anhydrases IV, IX, and XIV in the Mouse Heart"

Article Title: Expression of Membrane-bound Carbonic Anhydrases IV, IX, and XIV in the Mouse Heart

Journal:

doi: 10.1369/jhc.6A7003.2006

Double-immunofluorescence staining of CA IV and SERCA2 in adult cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA IV/FITC. ( C ) Anti-mouse SERCA2/TRITC. ( D
Figure Legend Snippet: Double-immunofluorescence staining of CA IV and SERCA2 in adult cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA IV/FITC. ( C ) Anti-mouse SERCA2/TRITC. ( D

Techniques Used: Double Immunofluorescence Staining, Incubation

Double-immunofluorescence staining of CA IX and SERCA2 in adult cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA IX/FITC. ( C ) Anti-mouse SERCA2/TRITC. ( D
Figure Legend Snippet: Double-immunofluorescence staining of CA IX and SERCA2 in adult cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA IX/FITC. ( C ) Anti-mouse SERCA2/TRITC. ( D

Techniques Used: Double Immunofluorescence Staining, Incubation

Double-immunofluorescence staining of CA XIV and MCT-1 in cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CAXIV/FITC. ( C ) Anti-mouse MCT-1/TRITC. ( D ) Merge
Figure Legend Snippet: Double-immunofluorescence staining of CA XIV and MCT-1 in cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CAXIV/FITC. ( C ) Anti-mouse MCT-1/TRITC. ( D ) Merge

Techniques Used: Double Immunofluorescence Staining, Incubation

Double immunofluorescence staining of CA IX and MCT-1 in cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA IX/FITC. ( C ) Anti-mouse MCT-1/TRITC. ( D ) Merge of
Figure Legend Snippet: Double immunofluorescence staining of CA IX and MCT-1 in cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA IX/FITC. ( C ) Anti-mouse MCT-1/TRITC. ( D ) Merge of

Techniques Used: Double Immunofluorescence Staining, Incubation

Double-immunofluorescence staining of CA XIV and anti-SERCA2 in adult cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA XIV/FITC. ( C ) Anti-mouse SERCA2/TRITC.
Figure Legend Snippet: Double-immunofluorescence staining of CA XIV and anti-SERCA2 in adult cardiomyocytes of mouse. ( A-D ) Cardiomyocytes of wild-type mouse. ( A ) Incubation with preimmune serum and anti-rabbit IgG/FITC. ( B ) Anti-mouse CA XIV/FITC. ( C ) Anti-mouse SERCA2/TRITC.

Techniques Used: Double Immunofluorescence Staining, Incubation

14) Product Images from "Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation *"

Article Title: Cbl-b Is a Novel Physiologic Regulator of Glycoprotein VI-dependent Platelet Activation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.080200

Association of Cbl-b with PLC γ2and BTK. A and B , human washed platelets were incubated with 200 ng/ml convulxin or vehicle as indicated. After preclearing, proteins were immunoprecipitated ( IP ) with either anti-Cbl-b ( A ) or anti-BTK ( B ). IgG was used as a negative control in both cases. Immunoprecipitates were analyzed by Western blotting using the indicated antibodies. C , platelet lysates were prepared as above and treated with agarose containing either GST or GST-Cbl-b. The agarose-bound material was analyzed by Western blotting. Blots were probed for either Cbl-b (GST-Cbl-b used for pull-down), PLCγ2, BTK, or Syk as labeled. Each experiment is representative of at least three similar experiments.
Figure Legend Snippet: Association of Cbl-b with PLC γ2and BTK. A and B , human washed platelets were incubated with 200 ng/ml convulxin or vehicle as indicated. After preclearing, proteins were immunoprecipitated ( IP ) with either anti-Cbl-b ( A ) or anti-BTK ( B ). IgG was used as a negative control in both cases. Immunoprecipitates were analyzed by Western blotting using the indicated antibodies. C , platelet lysates were prepared as above and treated with agarose containing either GST or GST-Cbl-b. The agarose-bound material was analyzed by Western blotting. Blots were probed for either Cbl-b (GST-Cbl-b used for pull-down), PLCγ2, BTK, or Syk as labeled. Each experiment is representative of at least three similar experiments.

Techniques Used: Planar Chromatography, Incubation, Immunoprecipitation, Negative Control, Western Blot, Labeling

15) Product Images from "Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease"

Article Title: Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease

Journal: EMBO Molecular Medicine

doi: 10.1002/emmm.201000084

TG2 levels and activity influence the activity of the cytochrome c promoter A. A cytochrome c promoter–reporter construct that contains the proximal 326 bp of the cytochrome c promoter (-326-luc), including response elements for specificity protein 1 (SP1), cAMP (CRE) and nuclear respiratory factor 1 (NRF-1). B. Q7 and Q111 cells transfected with the -326-luc construct followed by various durations of serum starvation and restimulation (serum re-addition). A more robust response of cytochrome c promoter activity is induced within Q7 cells (white bars) than Q111 cells (grey bars) relative to control condition (C) of no serum starvation. These findings indicate that that homeostatic response to energetic stress is repressed in cells expressing mhtt. C. Co-transfection of a WT TG2 construct with the cytochrome c promoter–reporter greatly inhibited promoter activity in Q7 and Q111 cells compared to cells that were transfected with GFP and the cytochrome c promoter–reporter. Similarly, a TG2 construct that does not retain cross-linking activity (C277S) lost the ability to repress the cytochrome c promoter activity in Q7 or Q111 cells. WT and mutant TG2 were expressed at the same protein levels in these experiments. D. Inhibition of TG activity by ZDON (50 µM for 12 h) increased the cytochrome c -326 promoter–reporter activity in Q111 cells nearly fourfold over non-treated cells (-326-luc w ZDON). Over-expression of TG2 repressed the promoter activity (-326-luc w TG2). E, F. Chromatin immunoprecipitation assays showed that more TG2 is located at the cytochrome c promoter (E) and at the PGC-1α gene (F) in non-treated Q111 cells compared to non-treated Q7 cells. TG2 occupancy was sharply decreased in Q111 cells treated with ZDON in both cases. TG2 was found at the cytochrome c promoter of WT MEFs (TG2 +/+ MEF) but not in MEFs derived from TG2 −/− cells (TG2 −/− MEF), confirming the specificity of the TG2 antibody used for ChIP assays. The data representing each sample is normalized with respect to both IgG immunoprecipitations and input. * p
Figure Legend Snippet: TG2 levels and activity influence the activity of the cytochrome c promoter A. A cytochrome c promoter–reporter construct that contains the proximal 326 bp of the cytochrome c promoter (-326-luc), including response elements for specificity protein 1 (SP1), cAMP (CRE) and nuclear respiratory factor 1 (NRF-1). B. Q7 and Q111 cells transfected with the -326-luc construct followed by various durations of serum starvation and restimulation (serum re-addition). A more robust response of cytochrome c promoter activity is induced within Q7 cells (white bars) than Q111 cells (grey bars) relative to control condition (C) of no serum starvation. These findings indicate that that homeostatic response to energetic stress is repressed in cells expressing mhtt. C. Co-transfection of a WT TG2 construct with the cytochrome c promoter–reporter greatly inhibited promoter activity in Q7 and Q111 cells compared to cells that were transfected with GFP and the cytochrome c promoter–reporter. Similarly, a TG2 construct that does not retain cross-linking activity (C277S) lost the ability to repress the cytochrome c promoter activity in Q7 or Q111 cells. WT and mutant TG2 were expressed at the same protein levels in these experiments. D. Inhibition of TG activity by ZDON (50 µM for 12 h) increased the cytochrome c -326 promoter–reporter activity in Q111 cells nearly fourfold over non-treated cells (-326-luc w ZDON). Over-expression of TG2 repressed the promoter activity (-326-luc w TG2). E, F. Chromatin immunoprecipitation assays showed that more TG2 is located at the cytochrome c promoter (E) and at the PGC-1α gene (F) in non-treated Q111 cells compared to non-treated Q7 cells. TG2 occupancy was sharply decreased in Q111 cells treated with ZDON in both cases. TG2 was found at the cytochrome c promoter of WT MEFs (TG2 +/+ MEF) but not in MEFs derived from TG2 −/− cells (TG2 −/− MEF), confirming the specificity of the TG2 antibody used for ChIP assays. The data representing each sample is normalized with respect to both IgG immunoprecipitations and input. * p

Techniques Used: Activity Assay, Construct, Transfection, Expressing, Cotransfection, Mutagenesis, Inhibition, Over Expression, Chromatin Immunoprecipitation, Pyrolysis Gas Chromatography, Derivative Assay

16) Product Images from "Genomic characterization of Wilms' tumor suppressor 1 targets in nephron progenitor cells during kidney development"

Article Title: Genomic characterization of Wilms' tumor suppressor 1 targets in nephron progenitor cells during kidney development

Journal: Development (Cambridge, England)

doi: 10.1242/dev.045732

Reduced WT1 expression and arrested development in WT1 morphant kidney explants. ( A , A′ , C , C′ ) E12.5 kidney explants cultured for 24 hours in media alone (A,A′) or in 10 μM control morpholino (cntl MO; C,C′) exhibit strong WT1 (red) expression in the cap of nephron progenitors (dashed outlines) surrounding the tip of the ureteric bud (green), as well as in epithelialized nephrogenic structures (arrows). Arrowheads denote regions shown in high magnification in adjacent panels. ( B , B′ ) Background auto-fluorescence in negative control explants incubated with rabbit IgG. ( D , D′ ) WT1 expression is markedly reduced in the cap region and moderately reduced in epithelialized nephrogenic structures of explants treated with 10 μM WT1 MO. The number of WT1-expressing nephrogenic tubules is also reduced in these explants. ( E , E′ ) Explants treated with 20 μM cntl MO exhibit moderate reductions in ureteric bud branching compared with explants cultured in media alone but continue to express WT1 in nephron progenitor cells and form WT1-expressing epithelialized nephrogenic structures. ( F , F′ ) By contrast, WT1 expression is not detected in explants treated with 20 μM WT1 MO, and explants do not undergo growth or ureteric bud branching.
Figure Legend Snippet: Reduced WT1 expression and arrested development in WT1 morphant kidney explants. ( A , A′ , C , C′ ) E12.5 kidney explants cultured for 24 hours in media alone (A,A′) or in 10 μM control morpholino (cntl MO; C,C′) exhibit strong WT1 (red) expression in the cap of nephron progenitors (dashed outlines) surrounding the tip of the ureteric bud (green), as well as in epithelialized nephrogenic structures (arrows). Arrowheads denote regions shown in high magnification in adjacent panels. ( B , B′ ) Background auto-fluorescence in negative control explants incubated with rabbit IgG. ( D , D′ ) WT1 expression is markedly reduced in the cap region and moderately reduced in epithelialized nephrogenic structures of explants treated with 10 μM WT1 MO. The number of WT1-expressing nephrogenic tubules is also reduced in these explants. ( E , E′ ) Explants treated with 20 μM cntl MO exhibit moderate reductions in ureteric bud branching compared with explants cultured in media alone but continue to express WT1 in nephron progenitor cells and form WT1-expressing epithelialized nephrogenic structures. ( F , F′ ) By contrast, WT1 expression is not detected in explants treated with 20 μM WT1 MO, and explants do not undergo growth or ureteric bud branching.

Techniques Used: Expressing, Cell Culture, Fluorescence, Negative Control, Incubation

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Co-Immunoprecipitation Assay:

Article Title: Coordinate Regulation of TET2 and EBNA2 Controls the DNA Methylation State of Latent Epstein-Barr Virus
Article Snippet: .. Rabbit IgG (Santa Cruz Biotechnology sc-2027), rabbit anti-TET2 (EMD Millipore ABE364), Myc (Cell Signaling 2278), mouse IgG (Santa Cruz Biotechnology sc-2025), or mouse anti-FLAG M2 (Sigma F1804) was used in co-IP. .. Antibodies used for Western blotting were rabbit polyclonal RBP-jκ (Abcam AB25949), TET2 (Proteintech 21207-1-AP), TET3 (GeneTex GTX121453), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Cell Signaling catalog number 2118), Myc (Cell Signaling catalog number 2278); rabbit polyclonal anti-Zta was generated from full-length Zta at Pocono Rabbit Farms; also used were rat monoclonal anti-EBNA2 (EMD Millipore MABE8), mouse monoclonal anti-TET2 (EMD Millipore MABE462), LMP1 (Dako M0897), EA-D (Abcam ab49668), sheep polyclonal anti-EBNA3C (Exalpha F125P), actin-peroxidase antibody (Sigma A3854), FLAG-peroxidase antibody (Sigma A8592).

Immunoprecipitation:

Article Title: Inflammation and Gli2 Suppress Gastrin Gene Expression in a Murine Model of Antral Hyperplasia
Article Snippet: .. For the “input,” 1% of the lysate was removed for PCR analysis and the remainder was used for immunoprecipitation overnight at 4°C with either rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZBP-89 or anti-GLI2 antibodies (Abcam, Cambridge, MA). .. The crosslinking was reversed and DNA was recovered using QIAquick PCR Purification Kit (Qiagen, Valencia, CA).

Article Title: Notch Signaling Pathway Enhances Bone Morphogenetic Protein 2 (BMP2) Responsiveness of Msx2 Gene to Induce Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells *
Article Snippet: .. The cross-linked DNA/protein extracts were diluted with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mmol/liter EDTA, 167 mmol/liter NaCl, 16.7 mmol/liter Tris-HCl, and protease inhibitors) and immunoprecipitated with 5 μg of normal rabbit IgG (Santa Cruz Biotechnology), anti-FLAG, or anti-RBPJk antibodies (sc-271128; Santa Cruz Biotechnology) overnight at 4 °C. .. Protein A-Sepharose beads were added to the supernatant, and the mixture was incubated for 1 h. The beads were washed sequentially with TSE I buffer (0.1% SDS, 1% Triton X-100, 2 mmol/liter EDTA, 150 mmol/liter NaCl, and 20 mmol/liter Tris-HCl), TSE II buffer (0.1% SDS, 1% Triton X-100, 2 mmol/liter EDTA, 500 mmol/liter NaCl, and 20 mmol/liter Tris-HCl), TSE III buffer (0.25 mol/liter LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mmol/liter EDTA, and 10 mmol/liter Tris-HCl), and TE buffer (10 mmol/liter Tris-HCl and 1 mmol/liter EDTA) twice.

Article Title: O-GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41
Article Snippet: .. Controls for immunoprecipitation specificities were performed with rabbit IgG (Santa Cruz Biotechnology). .. Antibody-bound proteins were recovered by rocking samples with 20 μl of Sepharose-labeled protein A/G (GE Healthcare, Buc, France) for 1 h at 4°C.

Article Title: GIV/Girdin Links Vascular Endothelial Growth Factor Signaling to Akt Survival Signaling in Podocytes Independent of Nephrin
Article Snippet: .. For immunoprecipitation, rat glomerular lysates (1 mg total protein) or mouse podocyte lysates (500 µ g total protein) were incubated 3 hours or overnight at 4°C with 2 µ l anti-VEGFR2 or anti-G α i3 or rabbit IgG (Santa Cruz Biotechnology) in Triton X-100 lysis buffer. .. Protein A magnetic beads (EMD Millipore) were added and incubated at 4°C for an additional 1 hour.

Incubation:

Article Title: Defects in Translational Regulation Mediated by the ? Subunit of Eukaryotic Initiation Factor 2 Inhibit Antiviral Activity and Facilitate the Malignant Transformation of Human Fibroblasts
Article Snippet: .. Whole-cell lysate was added to low-salt buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, 0.1% β-mercaptoethanol) and precleared by incubation with 50 μl of protein G agarose (Invitrogen Corp., Carlsbad, Calif.) and 2.5 μl of normal rabbit immunoglobulin G (Santa Cruz Biotechnology) for 1 h at 4°C. .. Protein G was removed by centrifugation, and an additional 50 μl of protein G slurry was added along with 5 μl of rabbit polyclonal anti-FLAG antiserum (Sigma).

Article Title: The p53-induced factor Ei24 inhibits nuclear import through an importin β–binding–like domain
Article Snippet: .. Precleared lysate was added to 10 µg anti-Ei24 (Sigma-Aldrich) or rabbit IgG (Santa Cruz Biotechnology, Inc.) antibody and 50 µl Protein A/G plus agarose slurry (Santa Cruz Biotechnology, Inc.), and incubated overnight at 4°C. ..

Article Title: GIV/Girdin Links Vascular Endothelial Growth Factor Signaling to Akt Survival Signaling in Podocytes Independent of Nephrin
Article Snippet: .. For immunoprecipitation, rat glomerular lysates (1 mg total protein) or mouse podocyte lysates (500 µ g total protein) were incubated 3 hours or overnight at 4°C with 2 µ l anti-VEGFR2 or anti-G α i3 or rabbit IgG (Santa Cruz Biotechnology) in Triton X-100 lysis buffer. .. Protein A magnetic beads (EMD Millipore) were added and incubated at 4°C for an additional 1 hour.

Polymerase Chain Reaction:

Article Title: Inflammation and Gli2 Suppress Gastrin Gene Expression in a Murine Model of Antral Hyperplasia
Article Snippet: .. For the “input,” 1% of the lysate was removed for PCR analysis and the remainder was used for immunoprecipitation overnight at 4°C with either rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-ZBP-89 or anti-GLI2 antibodies (Abcam, Cambridge, MA). .. The crosslinking was reversed and DNA was recovered using QIAquick PCR Purification Kit (Qiagen, Valencia, CA).

Lysis:

Article Title: GIV/Girdin Links Vascular Endothelial Growth Factor Signaling to Akt Survival Signaling in Podocytes Independent of Nephrin
Article Snippet: .. For immunoprecipitation, rat glomerular lysates (1 mg total protein) or mouse podocyte lysates (500 µ g total protein) were incubated 3 hours or overnight at 4°C with 2 µ l anti-VEGFR2 or anti-G α i3 or rabbit IgG (Santa Cruz Biotechnology) in Triton X-100 lysis buffer. .. Protein A magnetic beads (EMD Millipore) were added and incubated at 4°C for an additional 1 hour.

Chromatin Immunoprecipitation:

Article Title: EBNA1 binding and epigenetic regulation of gastrokine tumor suppressor genes in gastric carcinoma cells
Article Snippet: .. The following antibodies were used for ChIP assays: rabbit anti-EBNA1 (305/10 wk), anti- rabbit IgG (Santa Cruz Biotechnology, sc-2027). .. Anti-Actin HRP (Sigma, A3854) and rabbit anti-EBNA1 (305/10 wk) were used for Western Blotting.

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    Santa Cruz Biotechnology fitc conjugated donkey anti rabbit igg
    Immunofluorescence staining for detection of caspase 3 expression in isolated fetal rat lung fibroblasts. Isolated fetal rat lung fibroblasts were exposed to CSE (15% v/v) for three hours. Immunofluorescence was performed using caspase 3 rabbit polyclonal <t>IGg</t> antibody. Caspase 3 was visualized using donkey anti-rabbit <t>IGg-FITC</t> (green fluorescence) and counter stained with Hoescht 33342 (nuclear staining - blue). Image (A) shows controls not exposed to CSE. Image (B) shows expression of caspase 3 (green) primarily localized in the cytoplasm. Image (C) shows an enlarged image of a single cell.
    Fitc Conjugated Donkey Anti Rabbit Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti ubiad1 igg
    SCD-associated <t>UBIAD1</t> (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments ( A and B ). UBIAD1 − /pCDNA3.1, UBIAD1 − /pMyc-UBIAD1 (WT), and UBIAD1 − /pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 10 5 cells per 60-mm dish in medium A containing 10% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4 . After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presenc e of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with <t>polyclonal</t> anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with <t>IgG-A9</t> (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1. DOI: http://dx.doi.org/10.7554/eLife.05560.017
    Rabbit Polyclonal Anti Ubiad1 Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Brightness was quantified by image analysis software (Image Pro-Plus ver. 7.0) was measured (upper left). Tissue microarray analysis of 60 cases. β -actin was used as a positive control and normal rabbit <t>IgG</t> as a negative control (Gremlin, <t>FITC;</t> CD34, PI).
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    Santa Cruz Biotechnology rabbit polyclonal igg anti nos2
    Benznidazole inhibits inflammatory mediators in cultured cardiomyocytes. Cardiomyocytes were treated with different concentrations of Bzl for 30 min and then with 10 mg/L LPS for 48 h. <t>NOS2</t> expression was determined by Western blot with a specific antibody and normalized against α-actin. NO levels were quantified by the Griess reaction in culture supernatants (A). Cardiomyocytes were pre-treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 48 h. NOS2 expression was detected by immunofluorescence with a rabbit <t>polyclonal</t> antibody specific for NOS2 and a secondary FITC-labelled anti-rabbit <t>IgG.</t> Cells nuclei were stained with 300 nM DAPI. Representative microphotographs (400×) are shown (B). Cardiomyocytes were pre-treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 4 h. IL-6 and IL-1β mRNA levels were analysed by RT-qPCR and normalized against 18S rRNA (C). Scale bar: 10 μm. Results are expressed as mean of 3 independent experiments (3 replicates/treatment) ± SEM. * P
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    Immunofluorescence staining for detection of caspase 3 expression in isolated fetal rat lung fibroblasts. Isolated fetal rat lung fibroblasts were exposed to CSE (15% v/v) for three hours. Immunofluorescence was performed using caspase 3 rabbit polyclonal IGg antibody. Caspase 3 was visualized using donkey anti-rabbit IGg-FITC (green fluorescence) and counter stained with Hoescht 33342 (nuclear staining - blue). Image (A) shows controls not exposed to CSE. Image (B) shows expression of caspase 3 (green) primarily localized in the cytoplasm. Image (C) shows an enlarged image of a single cell.

    Journal: Tobacco Induced Diseases

    Article Title: Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke induced alterations

    doi: 10.1186/1617-9625-11-25

    Figure Lengend Snippet: Immunofluorescence staining for detection of caspase 3 expression in isolated fetal rat lung fibroblasts. Isolated fetal rat lung fibroblasts were exposed to CSE (15% v/v) for three hours. Immunofluorescence was performed using caspase 3 rabbit polyclonal IGg antibody. Caspase 3 was visualized using donkey anti-rabbit IGg-FITC (green fluorescence) and counter stained with Hoescht 33342 (nuclear staining - blue). Image (A) shows controls not exposed to CSE. Image (B) shows expression of caspase 3 (green) primarily localized in the cytoplasm. Image (C) shows an enlarged image of a single cell.

    Article Snippet: After four washes with PBS cells were incubated with secondary anti body, FITC conjugated donkey anti-rabbit IgG which recognizes rabbit IgG by immunofluorescence staining (Santa Cruz).

    Techniques: Immunofluorescence, Staining, Expressing, Isolation, Fluorescence

    Immunofluorescence staining for detection of caspase 3 expression in rat PDL cells. Rat PDL cells were exposed to CSE (15% v/v) for three hours. Immunofluorescence was performed using caspase 3 rabbit polyclonal IGg antibody. Caspase 3 was visualized using donkey anti-rabbit IGg-FITC (green fluorescence) and counter stained with Hoescht 33342 (nuclear staining - blue). Image (A) shows controls not exposed to CSE. Image (B) shows expression of caspase 3 (green) primarily localized in the cytoplasm. Image (C) shows enlarged image of a single cell.

    Journal: Tobacco Induced Diseases

    Article Title: Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke induced alterations

    doi: 10.1186/1617-9625-11-25

    Figure Lengend Snippet: Immunofluorescence staining for detection of caspase 3 expression in rat PDL cells. Rat PDL cells were exposed to CSE (15% v/v) for three hours. Immunofluorescence was performed using caspase 3 rabbit polyclonal IGg antibody. Caspase 3 was visualized using donkey anti-rabbit IGg-FITC (green fluorescence) and counter stained with Hoescht 33342 (nuclear staining - blue). Image (A) shows controls not exposed to CSE. Image (B) shows expression of caspase 3 (green) primarily localized in the cytoplasm. Image (C) shows enlarged image of a single cell.

    Article Snippet: After four washes with PBS cells were incubated with secondary anti body, FITC conjugated donkey anti-rabbit IgG which recognizes rabbit IgG by immunofluorescence staining (Santa Cruz).

    Techniques: Immunofluorescence, Staining, Expressing, Fluorescence

    SCD-associated UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments ( A and B ). UBIAD1 − /pCDNA3.1, UBIAD1 − /pMyc-UBIAD1 (WT), and UBIAD1 − /pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 10 5 cells per 60-mm dish in medium A containing 10% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4 . After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presenc e of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1. DOI: http://dx.doi.org/10.7554/eLife.05560.017

    Journal: eLife

    Article Title: The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    doi: 10.7554/eLife.05560

    Figure Lengend Snippet: SCD-associated UBIAD1 (N102S) resists geranylgeraniol-mediated displacement from HMG CoA reductase in two independent experiments ( A and B ). UBIAD1 − /pCDNA3.1, UBIAD1 − /pMyc-UBIAD1 (WT), and UBIAD1 − /pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 at a density of 4 × 10 5 cells per 60-mm dish in medium A containing 10% FCS. On day 3, cells were depleted of sterols as described in the legend to Figure 4 . After 16 hr at 37°C, cells received the identical medium containing 1 µg/ml 25-HC in the absence or presenc e of the indicated concentration of geranylgeraniol. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal anti-reductase antibodies. Aliquots of the precipitated material and the lysates were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), and anti-calnexin IgG. Proteins corresponding to immunoprecipitated UBIAD1 were quantified using ImageJ software. The intensities of these signals in the absence of geranylgeraniol were arbitrarily set as 1. DOI: http://dx.doi.org/10.7554/eLife.05560.017

    Article Snippet: Primary antibodies used for immunoblot analysis included: mouse monoclonal anti-T7 Tag IgG and anti-HSV Tag (Novagen, Darmstadt, Germany); IgG-A9, a mouse monoclonal antibody against the catalytic domain of reductase ( ); rabbit polyclonal anti-UBIAD1 IgG, mouse monoclonals IgG-H8 against UBIAD1, IgG-F2 against human peroxiredoxin-4, IgG-F3 against human progesterone receptor membrane component 2 (PGRMC2), IgG-C6 against human ERGIC-53, IgG-ANNEX 5E4/1 against human annexin I, and IgG-Y20 against human lamina-associated peptide-2 (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal anti-calnexin IgG (Novus Biologicals, Littleton, CO); and IgG-17H1, a mouse monoclonal antibody against human Insig-1.

    Techniques: Concentration Assay, Immunoprecipitation, SDS Page, Software

    The proteasome inhibitor MG-132 and geranylgeraniol inhibit sterol-induced binding of UBIAD1 to HMG CoA reductase. SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3 . Following sterol-depletion, cells received medium A containing 10% NC-LPPS, 10 µM compactin, 50 µM mevalonate in the absence or presence of 10 µM MG-132 for 1 hr at 37°C, followed by treatment in the absence or presence of 1 µg/ml 25-HC ( A ) or the indicated concentration of 25-HC in the absence or presence of 15 µM geranylgeraniol ( B ). Following incubation for 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against reductase. Aliquots of the resulting immunoprecipitates and lysates were subjected to immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG. DOI: http://dx.doi.org/10.7554/eLife.05560.009

    Journal: eLife

    Article Title: The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    doi: 10.7554/eLife.05560

    Figure Lengend Snippet: The proteasome inhibitor MG-132 and geranylgeraniol inhibit sterol-induced binding of UBIAD1 to HMG CoA reductase. SV-589 cells were set up for experiments on day 0 and depleted of sterols on day 3 as described in the legend to Figure 3 . Following sterol-depletion, cells received medium A containing 10% NC-LPPS, 10 µM compactin, 50 µM mevalonate in the absence or presence of 10 µM MG-132 for 1 hr at 37°C, followed by treatment in the absence or presence of 1 µg/ml 25-HC ( A ) or the indicated concentration of 25-HC in the absence or presence of 15 µM geranylgeraniol ( B ). Following incubation for 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against reductase. Aliquots of the resulting immunoprecipitates and lysates were subjected to immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG. DOI: http://dx.doi.org/10.7554/eLife.05560.009

    Article Snippet: Primary antibodies used for immunoblot analysis included: mouse monoclonal anti-T7 Tag IgG and anti-HSV Tag (Novagen, Darmstadt, Germany); IgG-A9, a mouse monoclonal antibody against the catalytic domain of reductase ( ); rabbit polyclonal anti-UBIAD1 IgG, mouse monoclonals IgG-H8 against UBIAD1, IgG-F2 against human peroxiredoxin-4, IgG-F3 against human progesterone receptor membrane component 2 (PGRMC2), IgG-C6 against human ERGIC-53, IgG-ANNEX 5E4/1 against human annexin I, and IgG-Y20 against human lamina-associated peptide-2 (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal anti-calnexin IgG (Novus Biologicals, Littleton, CO); and IgG-17H1, a mouse monoclonal antibody against human Insig-1.

    Techniques: Binding Assay, Concentration Assay, Incubation, Immunoprecipitation

    RNA interference-mediated knockdown of UBAD1 alleviates requirement for geranylgeraniol in sterol-accelerated reductase degradation. SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 3 . Notably, the siRNA duplex targeting UBIAD1 (5′-UCUUGGAGCCGCAGGAUGUUU-3′, Dharmacon/ThermoScientfic) was distinct from that used in Figures 3, 6 . The sterol-depleted cells were then treated with medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate in the absence or presence of 1 µg/ml 25-HC and 20 µM geranylgeraniol (GGOH). Following incubation for 4 hr at 37°C, cells were harvested for subcellular fractionation. Aliquots of resulting membrane fractions (20 µg protein/lane) were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase) and IgG-H8 (against UBIAD1). DOI: http://dx.doi.org/10.7554/eLife.05560.013

    Journal: eLife

    Article Title: The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    doi: 10.7554/eLife.05560

    Figure Lengend Snippet: RNA interference-mediated knockdown of UBAD1 alleviates requirement for geranylgeraniol in sterol-accelerated reductase degradation. SV-589 cells were set up for experiments on day 0, transfected with the indicated siRNA duplexes on day 3, and depleted of sterols as described in the legend to Figure 3 . Notably, the siRNA duplex targeting UBIAD1 (5′-UCUUGGAGCCGCAGGAUGUUU-3′, Dharmacon/ThermoScientfic) was distinct from that used in Figures 3, 6 . The sterol-depleted cells were then treated with medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate in the absence or presence of 1 µg/ml 25-HC and 20 µM geranylgeraniol (GGOH). Following incubation for 4 hr at 37°C, cells were harvested for subcellular fractionation. Aliquots of resulting membrane fractions (20 µg protein/lane) were subjected to SDS-PAGE and immunoblot analysis was carried out with IgG-A9 (against reductase) and IgG-H8 (against UBIAD1). DOI: http://dx.doi.org/10.7554/eLife.05560.013

    Article Snippet: Primary antibodies used for immunoblot analysis included: mouse monoclonal anti-T7 Tag IgG and anti-HSV Tag (Novagen, Darmstadt, Germany); IgG-A9, a mouse monoclonal antibody against the catalytic domain of reductase ( ); rabbit polyclonal anti-UBIAD1 IgG, mouse monoclonals IgG-H8 against UBIAD1, IgG-F2 against human peroxiredoxin-4, IgG-F3 against human progesterone receptor membrane component 2 (PGRMC2), IgG-C6 against human ERGIC-53, IgG-ANNEX 5E4/1 against human annexin I, and IgG-Y20 against human lamina-associated peptide-2 (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal anti-calnexin IgG (Novus Biologicals, Littleton, CO); and IgG-17H1, a mouse monoclonal antibody against human Insig-1.

    Techniques: Transfection, Incubation, Fractionation, SDS Page

    Schnyder corneal dystrophy (SCD)-associated N102S mutant of UBIAD1 blocks sterol-accelerated ERAD of full-length HMG CoA reductase. SV-589 cells were set up for experiments on day 0 at 4 × 10 5 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red-T7 in the absence or presence of the indicated concentration of wild type ( A ) or N102S ( B ) versions of pCMV-Myc-UBIAD1 as described in ‘Materials and methods’. 4 hr after transfection, cells received a direct addition of medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate (final concentrations). Following incubation for 16 hr at 37°C, cells were treated with identical medium in the absence or presence of 1 µg/ml 25-HC plus 20 µM geranylgeraniol (GGOH) as indicated. After 4 hr at 37°C, cells were harvested and subjected to subcellular fractionation. Aliquots of resulting membrane fractions were then subjected to SDS-PAGE and immunoblot analysis was carried out with anti-T7 IgG (against reductase), IgG-9E10 (against UBIAD1 and Insgi-1), and anti-calnexin IgG. Asterisks denote a non-specific cross-reactive band. Proteins corresponding to reductase in ( A and B ) were quantified using ImageJ software. The intensities of these signals in the absence of 25-HC plus geranylgeraniol were arbitrarily set as 1. DOI: http://dx.doi.org/10.7554/eLife.05560.015

    Journal: eLife

    Article Title: The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    doi: 10.7554/eLife.05560

    Figure Lengend Snippet: Schnyder corneal dystrophy (SCD)-associated N102S mutant of UBIAD1 blocks sterol-accelerated ERAD of full-length HMG CoA reductase. SV-589 cells were set up for experiments on day 0 at 4 × 10 5 cells per 60-mm dish in medium A containing 10% FCS. On day 1, cells were transfected with 3 µg/dish of pCMV-HMG-Red-T7 in the absence or presence of the indicated concentration of wild type ( A ) or N102S ( B ) versions of pCMV-Myc-UBIAD1 as described in ‘Materials and methods’. 4 hr after transfection, cells received a direct addition of medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate (final concentrations). Following incubation for 16 hr at 37°C, cells were treated with identical medium in the absence or presence of 1 µg/ml 25-HC plus 20 µM geranylgeraniol (GGOH) as indicated. After 4 hr at 37°C, cells were harvested and subjected to subcellular fractionation. Aliquots of resulting membrane fractions were then subjected to SDS-PAGE and immunoblot analysis was carried out with anti-T7 IgG (against reductase), IgG-9E10 (against UBIAD1 and Insgi-1), and anti-calnexin IgG. Asterisks denote a non-specific cross-reactive band. Proteins corresponding to reductase in ( A and B ) were quantified using ImageJ software. The intensities of these signals in the absence of 25-HC plus geranylgeraniol were arbitrarily set as 1. DOI: http://dx.doi.org/10.7554/eLife.05560.015

    Article Snippet: Primary antibodies used for immunoblot analysis included: mouse monoclonal anti-T7 Tag IgG and anti-HSV Tag (Novagen, Darmstadt, Germany); IgG-A9, a mouse monoclonal antibody against the catalytic domain of reductase ( ); rabbit polyclonal anti-UBIAD1 IgG, mouse monoclonals IgG-H8 against UBIAD1, IgG-F2 against human peroxiredoxin-4, IgG-F3 against human progesterone receptor membrane component 2 (PGRMC2), IgG-C6 against human ERGIC-53, IgG-ANNEX 5E4/1 against human annexin I, and IgG-Y20 against human lamina-associated peptide-2 (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal anti-calnexin IgG (Novus Biologicals, Littleton, CO); and IgG-17H1, a mouse monoclonal antibody against human Insig-1.

    Techniques: Mutagenesis, Transfection, Concentration Assay, Incubation, Fractionation, SDS Page, Software

    Specificity of sterol-dependent UBIAD1-HMG CoA reductase association. SV-589 cells were set up on day 0 at 1 × 10 5 cells per 100-mm dish in medium A containing 10% FCS. On day 3, cells were transfected with siRNAs targeting mRNAs encoding GFP, Insig-1 and Insig-2, UBIAD1, or reductase as indicated and described in ‘Materials and methods’. Cells transfected with siRNA duplexes against reductase received 200 mM mevalonate to provide essential nonsterol isoprenoids. On day 4, cells were depleted of sterols through incubation for 16 hr at 37°C in medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate. The cells then received identical medium in the absence or presence of 1 µg/ml 25-HC. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against either reductase ( A and B ) or UBIAD1 ( C ). The resulting precipitated material and lysates were subjected to SDS-PAGE and immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG. DOI: http://dx.doi.org/10.7554/eLife.05560.007

    Journal: eLife

    Article Title: The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    doi: 10.7554/eLife.05560

    Figure Lengend Snippet: Specificity of sterol-dependent UBIAD1-HMG CoA reductase association. SV-589 cells were set up on day 0 at 1 × 10 5 cells per 100-mm dish in medium A containing 10% FCS. On day 3, cells were transfected with siRNAs targeting mRNAs encoding GFP, Insig-1 and Insig-2, UBIAD1, or reductase as indicated and described in ‘Materials and methods’. Cells transfected with siRNA duplexes against reductase received 200 mM mevalonate to provide essential nonsterol isoprenoids. On day 4, cells were depleted of sterols through incubation for 16 hr at 37°C in medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate. The cells then received identical medium in the absence or presence of 1 µg/ml 25-HC. After 45 min at 37°C, cells were harvested, lysed, and immunoprecipitated with polyclonal antibodies against either reductase ( A and B ) or UBIAD1 ( C ). The resulting precipitated material and lysates were subjected to SDS-PAGE and immunoblot analysis with IgG-A9 (against reductase), IgG-H8 (against UBIAD1), IgG-17H1 (against Insig-1), and anti-calnexin IgG. DOI: http://dx.doi.org/10.7554/eLife.05560.007

    Article Snippet: Primary antibodies used for immunoblot analysis included: mouse monoclonal anti-T7 Tag IgG and anti-HSV Tag (Novagen, Darmstadt, Germany); IgG-A9, a mouse monoclonal antibody against the catalytic domain of reductase ( ); rabbit polyclonal anti-UBIAD1 IgG, mouse monoclonals IgG-H8 against UBIAD1, IgG-F2 against human peroxiredoxin-4, IgG-F3 against human progesterone receptor membrane component 2 (PGRMC2), IgG-C6 against human ERGIC-53, IgG-ANNEX 5E4/1 against human annexin I, and IgG-Y20 against human lamina-associated peptide-2 (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal anti-calnexin IgG (Novus Biologicals, Littleton, CO); and IgG-17H1, a mouse monoclonal antibody against human Insig-1.

    Techniques: Transfection, Incubation, Immunoprecipitation, SDS Page

    Geranylgeraniol, but not 25-HC or farnesol, stimulates translocation of transfected UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids. SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 10 5 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 10 4 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2 . DOI: http://dx.doi.org/10.7554/eLife.05560.011

    Journal: eLife

    Article Title: The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    doi: 10.7554/eLife.05560

    Figure Lengend Snippet: Geranylgeraniol, but not 25-HC or farnesol, stimulates translocation of transfected UBIAD1 to the Golgi in cells deprived of sterol and nonsterol isoprenoids. SV-589/pMyc-UBIAD1 cells, a line of SV-589 cells that stably express Myc-UBIAD1, were generated as follows. SV-589 cells were set up on day 0 at a density of 7 × 10 5 cells per 100-mm dish in medium A supplemented with 10% FCS. On day 1, cells were transfected with 2 µg/dish of pCMV-Myc-UBIAD1 using FuGENE6 transfection reagent as described in ‘Materials and methods’. Following incubation for 16 hr at 37°C, cells were switched to medium A supplemented with 10% FCS and 700 µg/ml G418. Fresh medium as added every 2–3 days until colonies formed after 2 weeks. Individual colonies were isolated using cloning cylinders, and expression of Myc-UBIAD1 was determined by immunoblot analysis. Select colonies were expanded and then further purified by serial dilution in 96-well plates. Individual clones were screened by immunofluorescense using IgG-9E10 against the Myc epitope. For experiments, SV-589/pMyc-UBIAD1 cells were set up on day 0 at 7.5 × 10 4 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol, 30 µM farnesol, or 1 µg/ml 25-HC for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining and analysis as described in the legend to Figure 5—figure supplement 2 . DOI: http://dx.doi.org/10.7554/eLife.05560.011

    Article Snippet: Primary antibodies used for immunoblot analysis included: mouse monoclonal anti-T7 Tag IgG and anti-HSV Tag (Novagen, Darmstadt, Germany); IgG-A9, a mouse monoclonal antibody against the catalytic domain of reductase ( ); rabbit polyclonal anti-UBIAD1 IgG, mouse monoclonals IgG-H8 against UBIAD1, IgG-F2 against human peroxiredoxin-4, IgG-F3 against human progesterone receptor membrane component 2 (PGRMC2), IgG-C6 against human ERGIC-53, IgG-ANNEX 5E4/1 against human annexin I, and IgG-Y20 against human lamina-associated peptide-2 (Santa Cruz Biotechnology, Dallas, TX); rabbit polyclonal anti-calnexin IgG (Novus Biologicals, Littleton, CO); and IgG-17H1, a mouse monoclonal antibody against human Insig-1.

    Techniques: Translocation Assay, Transfection, Stable Transfection, Generated, Incubation, Isolation, Clone Assay, Expressing, Purification, Serial Dilution, Immunostaining

    Brightness was quantified by image analysis software (Image Pro-Plus ver. 7.0) was measured (upper left). Tissue microarray analysis of 60 cases. β -actin was used as a positive control and normal rabbit IgG as a negative control (Gremlin, FITC; CD34, PI).

    Journal: International Journal of Endocrinology

    Article Title: Gremlin, a Bone Morphogenetic Protein Antagonist, Is a Crucial Angiogenic Factor in Pituitary Adenoma

    doi: 10.1155/2015/834137

    Figure Lengend Snippet: Brightness was quantified by image analysis software (Image Pro-Plus ver. 7.0) was measured (upper left). Tissue microarray analysis of 60 cases. β -actin was used as a positive control and normal rabbit IgG as a negative control (Gremlin, FITC; CD34, PI).

    Article Snippet: Thereafter, sections were incubated with rabbit anti-human Gremlin polyclonal IgG antibody (1 : 200; Santa Cruz) for 60 min followed by incubation with FITC-conjugated bovine anti-rabbit IgG (1 : 100; Santa Cruz) for 60 min. After counterstaining with Meyer's hematoxylin, each section was mounted with mounting medium (Gel/Mount, Biomeda Corp, Foster City, CA).

    Techniques: Software, Microarray, Positive Control, Negative Control

    Benznidazole inhibits inflammatory mediators in cultured cardiomyocytes. Cardiomyocytes were treated with different concentrations of Bzl for 30 min and then with 10 mg/L LPS for 48 h. NOS2 expression was determined by Western blot with a specific antibody and normalized against α-actin. NO levels were quantified by the Griess reaction in culture supernatants (A). Cardiomyocytes were pre-treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 48 h. NOS2 expression was detected by immunofluorescence with a rabbit polyclonal antibody specific for NOS2 and a secondary FITC-labelled anti-rabbit IgG. Cells nuclei were stained with 300 nM DAPI. Representative microphotographs (400×) are shown (B). Cardiomyocytes were pre-treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 4 h. IL-6 and IL-1β mRNA levels were analysed by RT-qPCR and normalized against 18S rRNA (C). Scale bar: 10 μm. Results are expressed as mean of 3 independent experiments (3 replicates/treatment) ± SEM. * P

    Journal: International Journal for Parasitology: Drugs and Drug Resistance

    Article Title: Low-dose benznidazole treatment results in parasite clearance and attenuates heart inflammatory reaction in an experimental model of infection with a highly virulent Trypanosoma cruzi strain

    doi: 10.1016/j.ijpddr.2015.12.001

    Figure Lengend Snippet: Benznidazole inhibits inflammatory mediators in cultured cardiomyocytes. Cardiomyocytes were treated with different concentrations of Bzl for 30 min and then with 10 mg/L LPS for 48 h. NOS2 expression was determined by Western blot with a specific antibody and normalized against α-actin. NO levels were quantified by the Griess reaction in culture supernatants (A). Cardiomyocytes were pre-treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 48 h. NOS2 expression was detected by immunofluorescence with a rabbit polyclonal antibody specific for NOS2 and a secondary FITC-labelled anti-rabbit IgG. Cells nuclei were stained with 300 nM DAPI. Representative microphotographs (400×) are shown (B). Cardiomyocytes were pre-treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 4 h. IL-6 and IL-1β mRNA levels were analysed by RT-qPCR and normalized against 18S rRNA (C). Scale bar: 10 μm. Results are expressed as mean of 3 independent experiments (3 replicates/treatment) ± SEM. * P

    Article Snippet: For this purpose, rabbit polyclonal IgG anti-NOS2 or IgG anti-IκBα (Santa Cruz Biotechnology, CA, USA) and FITC-labelled goat anti-rabbit IgG (Sigma–Aldrich Co), were used at 1:200 dilutions (determined by titration).

    Techniques: Cell Culture, Expressing, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR

    Benznidazole at low concentrations also inhibits the NF-κB pathway. Cardiomyocytes were treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 30, 60 or 120 min. IκBα and p65 cytosolic expression were determined by Western blot with specific antibodies. Protein levels were normalized against α-actin. A representative result out of 3 experiments performed is shown (A). IκBα expression was detected by immunofluorescence with a polyclonal rabbit anti-IκBα antibody and a secondary FITC-labelled anti-rabbit IgG. Cells nuclei were stained with 300 nM DAPI. Representative microphotographs (400×) are shown (B). Scale bar: 10 μm.

    Journal: International Journal for Parasitology: Drugs and Drug Resistance

    Article Title: Low-dose benznidazole treatment results in parasite clearance and attenuates heart inflammatory reaction in an experimental model of infection with a highly virulent Trypanosoma cruzi strain

    doi: 10.1016/j.ijpddr.2015.12.001

    Figure Lengend Snippet: Benznidazole at low concentrations also inhibits the NF-κB pathway. Cardiomyocytes were treated with 15 μM Bzl for 30 min and then with 10 mg/L LPS for 30, 60 or 120 min. IκBα and p65 cytosolic expression were determined by Western blot with specific antibodies. Protein levels were normalized against α-actin. A representative result out of 3 experiments performed is shown (A). IκBα expression was detected by immunofluorescence with a polyclonal rabbit anti-IκBα antibody and a secondary FITC-labelled anti-rabbit IgG. Cells nuclei were stained with 300 nM DAPI. Representative microphotographs (400×) are shown (B). Scale bar: 10 μm.

    Article Snippet: For this purpose, rabbit polyclonal IgG anti-NOS2 or IgG anti-IκBα (Santa Cruz Biotechnology, CA, USA) and FITC-labelled goat anti-rabbit IgG (Sigma–Aldrich Co), were used at 1:200 dilutions (determined by titration).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining