erbb3 apc  (Miltenyi Biotec)

Journal: Bladder Cancer (Amsterdam, Netherlands)

Article Title: Molecular Correlates of In Vitro Responses to Dacomitinib and Afatinib in Bladder Cancer

doi: 10.3233/BLC-170144

Figure Lengend Snippet: Evaluation of afatinib sensitivity with Her2 or Her3 mutation

Article Snippet: The following antibodies were used: Phycoerythrin or fluorescein isothiocyanate labeled rat monoclonal antibody to human EGFR (ICR 10, Abcam, Eugene, OR); allophycocyanin (APC) labeled anti-HER2 (Becton Dickinson Biosciences, San Jose, CA); APC labeled anti-human HER3 (R&D Systems, Minneapolis, MN); APC labeled anti-human HER4 (R&D Systems, Minneapolis, MN), and isotype controls mouse IgG2A (R&D Systems, Minneapolis, MN) and mouse IgG1 (Miltenyi Biotech, San Diego, CA).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family

doi: 10.1371/journal.pone.0148711

Figure Lengend Snippet: For (A) and (B) the hepatoma cell lines Huh7 and Huh7.5 were cultured for 48 hours while primary human hepatocytes (PHH) were isolated and cultured for 24 hours. Subsequently (A) total mRNA was analysed for transcript abundance of EGFR, ErbB2, ErbB3 and ErbB4 by rtPCR and for (B) total protein lysates were prepared and protein expression of EGFR, ErbB2 and ErbB3 was analysed by immunoblot using specific antibodies. β-actin or GAPDH levels were determined as loading controls. For (C) to (E) Huh cell lines harbouring the subgenomic replicon of HCV genotype 1b and the respective control cell line Huh7 cells were cultured for 48 hours and thereafter for (C) total RNA and for (D and E) total protein extracts were prepared and analysed for the abundance of EGFR, ErbB2, ErbB3 and ErbB4 transcripts (C) or for the protein levels of ErbB3 (D) as well as EGFR and ErbB2 (E). Additionally, NS3 expression was assessed for control of replication and β-actin levels for loading control. For (F) Huh9-13 cells were cultured for 24 hours and subsequently treated with 2-C’-Methylcytidine as indicated. After an additional 48 hours total protein extracts were prepared and analysed for ErbB3 and NS3 protein levels by immunoblot. (G and H) Huh7.5 cells were infected with 1 MOI of the HCVcc strain JC1 or left un-infected for control. Total RNA or protein lysates were prepared 48 hours after infection for quantification of the ErbB3 transcript (G) or 72 hours after infection for abundance of the respective protein by immunoblot using antibodies specifically recognizing ErbB3, NS5A or β-actin (H). For (I) Huh7.5 cells were differentiated by DMSO as summarized in the Material and Methods section and infected with the JC1 virus or left uninfected for control. Four weeks after infection total protein extracts were prepared and ErbB3 protein abundance was determined. To prove the differentiation status of the respective cell models used, Huh7.5 cells were either cultured for 5 days without DMSO treatment (H) or for 4 weeks in the presence of DMSO (I). After the respective culture period cells were fixed with ice cold methanol and analysed by immunofluorescence and confocal laser scanning imaging for the Na + /K + ATPase as a basolateral marker (red) and for the presence of MRP2 which indicates polarization and formation of an apical compartment (green). Since MRP2 is only expressed in the apical membrane positive staining for MRP2 indicates cellular differentiation coinciding with polarization (compare immunofluorescence depicted in (H) with that in (I)). For (A), (C) and (G) semiquantitative rtPCR results were calculated using the ΔΔCT method and SDHA as control gene and for (A) data are presented as means + SEM of at least seven or more independent experiments. For (C) and (G) data are provided as fractions of the respective control cells which were set to one and are depicted as means + SEM of at least three independent experiments. The blots presented in (D) to (F), (H) and (I) were evaluated densitometrically and relative expression of ErbB3 (D,F,H and I), EGFR or ErbB2 (E) was normalized to β-actin and data are expressed as fractions of the normalized value of the respective control, which was set to one. Data are presented as means + SEM of at least three or more independent experiments. p ≤ 0.05 was considered to be significant.

Article Snippet: The anti Phycoerythrin-coupled EGFR antibody (#FAB10951P), the Allophycocyanin-coupled ErbB3/HER3 antibody (#FAB3481A) as well as the corresponding controls (Rat IgG2A Isotype Control Phycoerythrin Conjugated: #IC006P, Mouse IgG1 Isotype Control APC Conjugated: #IC002A) used for FACS were obtained from R&D Systems (Minneapolis, USA).

Techniques: Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Infection, Virus, Immunofluorescence, Imaging, Marker, Membrane, Staining, Cell Differentiation

Journal: PLoS ONE

Article Title: Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family

doi: 10.1371/journal.pone.0148711

Figure Lengend Snippet: (A) to (C) Huh 9–13 cells were transfected with control siRNA or NRG1-specific siRNA as outlined in the Materials and Methods section. 72 hours after transfection total RNA or protein was extracted and amounts of NRG1 and ErbB3 transcripts were quantified by rtPCR (A) as outlined in figure legend to and the Material and Methods section, whereas expression of the viral proteins NS5A and NS5B as well as of ErbB3 were determined by immunoblot (B). β-actin expression was assessed as loading control. (C) Knock-down of NRG1 expression was performed in Huh9-13 replicon cells using NRG1 specific siRNA or respective control siRNA as indicated and culture was continued for in total 72 hours after transfection. 10 μM Triciribine or DMSO that served as control were added 12 hours prior to the end of the total incubation period of 72 hours as depicted and total protein extracts were prepared to analyse the expression of ErbB3, NS3 and β-actin by immunoblot. (D) and (E) Huh7 cells were pre-treated with 25 ng/ml of the NRG1 isoform Heregulin 1β for the time periods indicated. Thereafter total RNA was isolated and the abundance of the ErbB3 transcript was assessed by rtPCR (D) while immunoblot was used to determine ErbB3 protein levels (E). For comparison, protein extracts from untreated Huh9-13 replicon cells were analysed in parallel. Results of at least 3 independent experiments are depicted. For (A) and (D) data were calculated as outlined in the legend to and data are presented as mean + SEM.

Article Snippet: The anti Phycoerythrin-coupled EGFR antibody (#FAB10951P), the Allophycocyanin-coupled ErbB3/HER3 antibody (#FAB3481A) as well as the corresponding controls (Rat IgG2A Isotype Control Phycoerythrin Conjugated: #IC006P, Mouse IgG1 Isotype Control APC Conjugated: #IC002A) used for FACS were obtained from R&D Systems (Minneapolis, USA).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Incubation, Isolation, Comparison

Journal: PLoS ONE

Article Title: Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family

doi: 10.1371/journal.pone.0148711

Figure Lengend Snippet: (A) Huh7 or Huh9-13 cells were treated with Mithramycin A at the concentrations indicated or left untreated for control. Four and 16 hours after addition of Mithramycin A, total RNA was isolated and NRG1 mRNA expression was quantified by rtPCR. Values were normalized to those obtained from the control cell line Huh7. (B) Huh9-13 cells were transfected with a Sp1-specific siRNA. 72 hours later total RNA was isolated and abundance of Sp1, NRG1 and ErbB3 mRNA was quantified by rtPCR (left, middle and right subpanel, respectively) as outlined in the figure legend to and the Material and Methods section. Values were normalized to those obtained with control siRNA-transfected cells. (C) Huh7.5 cells were transfected with control siRNA or Sp1-specific siRNA as indicated and 48 hours later cells were infected with the HCV strain JC1 using a MOI of 1 TCID 50 /cell. After additional 48 hours total RNA extracts were prepared and amounts of Sp1 and NRG1 RNA was determined by rtPCR. (D) Huh7 cells were transfected with a Sp1-encoding plasmid and 48 hours later total RNA was extracted and amounts of Sp1-, NRG1- and ErbB3-specific transcripts were quantified by rtPCR. Semiquantitative PCR results were calculated as outlined in the legend to and data are presented as mean + SEM.

Article Snippet: The anti Phycoerythrin-coupled EGFR antibody (#FAB10951P), the Allophycocyanin-coupled ErbB3/HER3 antibody (#FAB3481A) as well as the corresponding controls (Rat IgG2A Isotype Control Phycoerythrin Conjugated: #IC006P, Mouse IgG1 Isotype Control APC Conjugated: #IC002A) used for FACS were obtained from R&D Systems (Minneapolis, USA).

Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Infection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family

doi: 10.1371/journal.pone.0148711

Figure Lengend Snippet: (A) and (B) Huh9-13 replicon cells were analysed by FACS to determine surface expression of ErbB3 and EGFR (A) or of ErbB3 and ErbB2 (B). (C) and (D) Huh7 cells were transfected with ErbB3 specific siRNA or with control siRNA. 72 hours later cells were prepared for FACS analysis to determine the amounts of ErbB3 and EGFR (C) or of ErbB3 and ErbB2 (D) on the cell surface. Data were analysed using the FlowJo software against the corresponding isotype controls and analysis of EGFR (A and C) or ErbB2 (B and D) surface expression was gated to cells with low expression of ErbB3. Results correspond to the average of the receptors at the cell surface (in precent); error bars indicate SEM from at least 3 independent experiments.

Article Snippet: The anti Phycoerythrin-coupled EGFR antibody (#FAB10951P), the Allophycocyanin-coupled ErbB3/HER3 antibody (#FAB3481A) as well as the corresponding controls (Rat IgG2A Isotype Control Phycoerythrin Conjugated: #IC006P, Mouse IgG1 Isotype Control APC Conjugated: #IC002A) used for FACS were obtained from R&D Systems (Minneapolis, USA).

Techniques: Expressing, Transfection, Software

Journal: PLoS ONE

Article Title: Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family

doi: 10.1371/journal.pone.0148711

Figure Lengend Snippet: (A) Huh7.5 cells were pretreated with 25 ng/ml of the NRG1 homologue HRG-1β as indicated and subsequently infected with 1 MOI of the HCVcc strain JC1. Seventy-two hours after infection total RNA was prepared and abundance of the HCV genome was determined by rtPCR. (B) Huh7.5 cells were transfected with control siRNA or ErbB3-specific siRNA as described in Materials and Methods. Forty-eight hours after transfection cells were infected with the HCVcc strain JC1 using 1 MOI. After another 48 hours total RNA was extracted and analysed for the abundance of HCV RNA genomes and of succinate dehydrogenase complex subunit A (SDHA) by rtPCR. Semi-quantitative PCR results were calculated as outlined in the legend to from at least three independent experiments and are presented means + SEM.

Article Snippet: The anti Phycoerythrin-coupled EGFR antibody (#FAB10951P), the Allophycocyanin-coupled ErbB3/HER3 antibody (#FAB3481A) as well as the corresponding controls (Rat IgG2A Isotype Control Phycoerythrin Conjugated: #IC006P, Mouse IgG1 Isotype Control APC Conjugated: #IC002A) used for FACS were obtained from R&D Systems (Minneapolis, USA).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Transfection, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Hepatitis C Virus Activates a Neuregulin-Driven Circuit to Modify Surface Expression of Growth Factor Receptors of the ErbB Family

doi: 10.1371/journal.pone.0148711

Figure Lengend Snippet: HCV up-regulates production of the ErbB3 ligand NRG1 (see also ) in its host cell by a mechanism that involves Sp1 (see also ). NRG1 in turn mediates down-regulation of ErbB3 transcript expression via an Akt-dependent pathway, which is also ligand-independently activated by HCV itself, and also of ErbB3-protein levels (see also ). Points which are not part of the scheme are that down-regulation of ErbB3 expression leads to an up-regulation of EGFR and ErbB2 expression by yet unknown mechanisms and promotes viral replication and infectivity.

Article Snippet: The anti Phycoerythrin-coupled EGFR antibody (#FAB10951P), the Allophycocyanin-coupled ErbB3/HER3 antibody (#FAB3481A) as well as the corresponding controls (Rat IgG2A Isotype Control Phycoerythrin Conjugated: #IC006P, Mouse IgG1 Isotype Control APC Conjugated: #IC002A) used for FACS were obtained from R&D Systems (Minneapolis, USA).

Techniques: Expressing, Infection