Journal: International Journal of Molecular Sciences

Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

doi: 10.3390/ijms24021443

Figure Lengend Snippet: Clinicopathological characteristics of breast cancer patients.

Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

doi: 10.3390/ijms24021443

Figure Lengend Snippet: Co-expression of EGFR and HER2 correlates with poor survival in breast cancer patients. ( A ) Disease-free survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.0229). ( B ) Overall survival rates were compared between EGFR+/HER2-/ER- ( n = 92) and EGFR+/HER2+/ER- ( n = 23) patients ( p = 0.019).

Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

doi: 10.3390/ijms24021443

Figure Lengend Snippet: Co-expression of EGFR and HER2 promotes invasion and proliferation. ( A ) Transcript levels of EGFR and HER2 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Values were normalized to ACTB . ( B ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( C ) Invasion assays were performed using MDA-MB231 Vec and HER2-overexpressed cells as described in the method section. The scale bar represents 200 μm. ( D ) Cell cycle analysis was performed using MDA-MB231 Vec and HER2-overexpressed cells. * p < 0.05.

Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Cell Cycle Assay

Journal: International Journal of Molecular Sciences

Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

doi: 10.3390/ijms24021443

Figure Lengend Snippet: Cytokine secretion profile is altered by HER2 overexpression. ( A ) Human cytokine array was performed using conditioned culture media of MDA-MB231 Vec and HER2-overexpressed cells. ( B – E ) Transcript levels of CCL2 , CCL5 , IL-6 , and IL-8 in Vec and HER2-overexpressed MDA-MB231 cells were analyzed using RT-qPCR. Values were normalized to ACTB . * p < 0.05.

Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

Techniques: Over Expression, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

doi: 10.3390/ijms24021443

Figure Lengend Snippet: CCL2 expression is downregulated by neratinib. ( A , B ) Each cell line was treated with 5 μM neratinib for 24 h. ( A ) Transcript levels of CCL2 were analyzed using RT-qPCR. Values were normalized to ACTB . ( B ) ELISA was performed using conditioned culture media of Vec and HER2-overexpressed MDA-MB231 cells. ( C ) Levels of protein expression were analyzed using Western blotting with indicated antibodies. β-actin was used as a loading control. ( D ) ELISA was performed using conditioned culture media of MDA453 and BT474 cells transfected with scrambled or HER2-specific siRNA. * p < 0.05; ** p < 0.01; ϕ p < 0.05.

Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

doi: 10.3390/ijms24021443

Figure Lengend Snippet: CCL2 mediates motility of TAMs. ( A ) Immunofluorescence confocal microscopy was performed using M0 and M2-differentiated THP-1 cells. Anti-CD11b primary antibody and Alexa Fluor 488-conjugated secondary antibody were used for staining. The nucleus was stained with DAPI. ( B ) Upper, levels of CD-11b and CD163 protein expression were analyzed using Western blotting with whole cell lysates from M0 and M2-differentiated THP-1 cells with indicated antibodies. β-actin was used as a loading control. Lower, transcript levels of CD11b in M0 and M2-differentiated THP-1 cells were analyzed using RT-qPCR. Values were normalized to ACTB. ( C ) Migration assays were performed as described in the method section. ( D ) Cytokine arrays were performed using whole cell lysates of control and CCL2-treated M2-differentiated THP-1 cells. ( E ) Migration assays were performed with M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. The scale bar represents 200 μm. ( F ) RT-qPCR showing transcript levels of IL-8 and IL-1B in M2-differentiated THP-1 cells treated with conditioned culture media from MDA-MB231 Vec and HER2-overexpressed cells. Values were normalized to ACTB . * p < 0.05.

Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Expressing, Western Blot, Quantitative RT-PCR, Migration

Journal: International Journal of Molecular Sciences

Article Title: Tumorigenicity of EGFR- and/or HER2-Positive Breast Cancers Is Mediated by Recruitment of Tumor-Associated Macrophages

doi: 10.3390/ijms24021443

Figure Lengend Snippet: TAMs recruited into HER2-overexpressed tumors. ( A ) A schematic diagram illustrating the xenograft study in this figure: MDA-MB231 Vec or HER2-overexpressed cells were injected into the 2nd fat pad of NOD/SCID mice. Recruited TAMs were monitored after 12 weeks. ( B ) Recruited TAMs were analyzed using CD163 staining.

Article Snippet: Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p -EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p -HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000).

Techniques: Injection, Staining

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet: ERBB signaling constitutes the core of KRAS dependency (A) PPI network of the 30 genes generated with STRING and visualized with Cytoscape. Colors indicate log 2 FC (nodes’ size indicates absolute log 2 FC) in gene expression between 20 most mt KRAS-dependent and 20 most mt KRAS-independent cell lines. (B) Results from GO enrichment analysis of the 30 genes using ShinyGO. Only terms with FDR<0.01 are plotted. Expression of genes from each category was averaged among cell lines with smallest and highest KDS30 scores. ‘Any (L)’ represents mt KRAS cancer cell lines with 10 lowest KDS30 scores. ‘Any (H)’ represents mt KRAS cancer cell lines with 10 highest KDS30 scores. ‘Panc (L)’ and ‘Panc (H)’ represent mt KRAS pancreatic cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. ‘Lung (L)’ and ‘Lung (H)’ represent mt KRAS lung cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. (C and E) EGFR/ERBB2 (in blue), MEK (in red) and RAF (in green) inhibitors that were found to selectively kill high KDS30 over low KDS30 PAAD (in C) or LUAD (in E) cancer cells, considering max top 40 drugs. PRISM_1ry: primary PRISM Repurposing screen, PRISM_2ry: secondary PRISM Repurposing screen, CTRP: Cancer Therapeutics Response Portal v2. Y-axis: difference in mean FC or mean AUC, see main text. Only inhibitors of EGFR/ERBB2/MEK/RAF with p<0.05 are shown. (D and F) Results from drug sensitivity data analysis for mt KRAS pancreatic cancer cells included in the PRISM 2ry screen (D) and mt KRAS lung cancer cell lines included in the CTRP screen (F). Mean AUC of high KDS30 cells was subtracted from mean AUC of low KDS30 cells and plotted on X-axis. See main text for thresholds. Student’s t - test was applied to determine the statistical significance to observe the difference and plotted on Y-axis. MEK inhibitors are highlighted in red. EGFR/ERBB2 inhibitors are highlighted in blue. NERA: neratinib; SELU: selumetinib; TRAM: trametinib; PD31: PD-318088; AFAT: afatinib; GEFI: gefitinib; AZD8: AZD8931; DACO: dacomitinib; TUCA: tucatinib; CANE: canertinib; COBI: cobimetinib; AS70: AS-703026; OSIM: Osimertinib; ERLO: erlotinib . See also Figure S3 .

Article Snippet: The following antibodies were purchased from Cell Signaling Technology, Inc., Denver, USA: ERK (catalog no. 4695S), p -ERK (catalog no. 9101S), p -EGFR(Tyr-1068) (catalog no. 3777S), p-HER2/ErbB2 (Tyr 1221/1222) (catalog no. 2243S), cleaved Caspase-3 (catalog no. 9664S), cleaved PARP (catalog no. 9541S).

Techniques: Generated, Expressing

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet: mt KRAS addicted lung cancer lines are highly sensitive to the combination of EGFR/ERBB2 inhibitors with MEK inhibitors High KDS30 H358 and low KDS30 LU99 lung cancer cells were treated with vehicle control (Ctrl), an EGFR/ERBB2 inhibitor and a MEK inhibitor, either alone or in combination as indicated on each figure panel, for 48 h and processed for western blotting as described in . pEGFR, pErbB2, pERK: phosphorylated EGFR, ErbB2, and ERK, respectively. c-Casp3 and c-PARP: cleaved Casp3 and PARP, respectively. Actin: loading control. Single ∗ and double ∗∗ indicate gels each protein is blotted from. See also Figure S4 .

Article Snippet: The following antibodies were purchased from Cell Signaling Technology, Inc., Denver, USA: ERK (catalog no. 4695S), p -ERK (catalog no. 9101S), p -EGFR(Tyr-1068) (catalog no. 3777S), p-HER2/ErbB2 (Tyr 1221/1222) (catalog no. 2243S), cleaved Caspase-3 (catalog no. 9664S), cleaved PARP (catalog no. 9541S).

Techniques: Western Blot

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet:

Article Snippet: The following antibodies were purchased from Cell Signaling Technology, Inc., Denver, USA: ERK (catalog no. 4695S), p -ERK (catalog no. 9101S), p -EGFR(Tyr-1068) (catalog no. 3777S), p-HER2/ErbB2 (Tyr 1221/1222) (catalog no. 2243S), cleaved Caspase-3 (catalog no. 9664S), cleaved PARP (catalog no. 9541S).

Techniques: Recombinant, Expressing, CRISPR, Software

Journal: Journal of Cancer

Article Title: In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC

doi: 10.7150/jca.46320

Figure Lengend Snippet: A) Chemical structure of 18. 3-amino-3-(1-napthyl propionic acid) (Anapa) can have R or S configuration. Active compound has R configuration at Anapa. B) proposed binding site of 18 on C-terminal HER2 domain IV. C) Binding of 18 to HER2 and inhibition of EGFR-HER2 or HER2-HER3 dimerization and modulation of downstream signaling for cell proliferation by different pathways.

Article Snippet: Antibodies for the detection of total HER2 protein (t-HER2), phosphorylated HER2 protein (p-HER2), total AKT (t-AKT); (Cell Signaling Technology, phosphorylated AKT (P-Akt, S473); (Cell Signaling Technology) were used at 1:1000 dilution.

Techniques: Binding Assay, Inhibition

Journal: Journal of Cancer

Article Title: In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC

doi: 10.7150/jca.46320

Figure Lengend Snippet: Effect of 18 on phosphorylation of HER2 and Akt. A549 lung cancer cell lines were incubated with 18 and after 36 h, cells were washed and lysed. Western blot was performed to determine p-HER2 and p-Akt as well as total HER2 and Akt. (Blots were cropped and presented). Quantification of signal intensities were done by ImageJ). Lapatinib was used as positive control. A) Western blot of HER2, p-HER2, Akt, p-Akt. Equal loading of GAPDH was used for comparison. Experiments were repeated three times. Quantification of B) p-HER2 and C) p-Akt using densitometry. Band intensity was represented with scaling from GAPDH band intensity. * p< 0.05, ** p< 0.01.

Article Snippet: Antibodies for the detection of total HER2 protein (t-HER2), phosphorylated HER2 protein (p-HER2), total AKT (t-AKT); (Cell Signaling Technology, phosphorylated AKT (P-Akt, S473); (Cell Signaling Technology) were used at 1:1000 dilution.

Techniques: Incubation, Western Blot, Positive Control

Journal: Journal of Cancer

Article Title: In vivo studies of a peptidomimetic that targets EGFR dimerization in NSCLC

doi: 10.7150/jca.46320

Figure Lengend Snippet: Effect of 18 on phosphorylation of HER2 from lung tumor tissue sections of mice harvested at 35 th day. Frozen tumors were processed and Western blot was carried out. (Blots were cropped and presented). Quantification of signal intensities were done by ImageJ). A) Western blot images of HER-2, p-HER-2 and GAPDH. B) Quantitative analysis of Western blot images indicating reduction in phosphorylation of p-HER2 by 18 and lapatinib (10 mg/kg) compared to the control. C) Inhibition of EGFR dimerization in NSCLC tissues from mice by 18 (6 mg/kg) using PLA. Representative tissue sections from lungs of mice from different group of animals were subjected to PLA. Red fluorescence indicates dimerization of EGFR:HER2 (top panel) and HER2:HER3(bottom panel). Expanded regions are shown as insets. Note the reduction in the number of red fluorescence dots in 18 -treated lung tissue. Magnification 40×, size of scale bar 20 μm. Nuclei were stained with DAPI.

Article Snippet: Antibodies for the detection of total HER2 protein (t-HER2), phosphorylated HER2 protein (p-HER2), total AKT (t-AKT); (Cell Signaling Technology, phosphorylated AKT (P-Akt, S473); (Cell Signaling Technology) were used at 1:1000 dilution.

Techniques: Western Blot, Inhibition, Fluorescence, Staining

Journal: Drug Design, Development and Therapy

Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

doi: 10.2147/DDDT.S151511

Figure Lengend Snippet: Primer sequences used for the qPCR analysis

Article Snippet: The following antibodies and concentrations were used over night at 4°C; NRG1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-28916; 1:400), ErbB4 (Santa Cruz Biotechnology Inc., sc-283; 1:500), ErbB2 (Cell Signaling, Danvers, MA, USA, 2165; 1:1,500), p-ErbB4 (Tyr1056) (Santa Cruz Biotechnology Inc., sc33040; 1:300), p-ErbB2 (Tyr1248) (Cell Signaling, 2247; 1:1,500), p-Akt (Ser473) (Cell Signaling, 4060; 1:3,000), p-ERK (Thr202/Tyr204) (Cell Signaling, 4695; 1:2,000), caspase-3 (Abcam, Cambridge, UK, ab4051; 1:500), and β-actin (Proteintech, Rosemont, IL, USA, 66009-1-Ig; 1:4,000).

Techniques: Amplification

Journal: Drug Design, Development and Therapy

Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

doi: 10.2147/DDDT.S151511

Figure Lengend Snippet: Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.

Article Snippet: The following antibodies and concentrations were used over night at 4°C; NRG1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-28916; 1:400), ErbB4 (Santa Cruz Biotechnology Inc., sc-283; 1:500), ErbB2 (Cell Signaling, Danvers, MA, USA, 2165; 1:1,500), p-ErbB4 (Tyr1056) (Santa Cruz Biotechnology Inc., sc33040; 1:300), p-ErbB2 (Tyr1248) (Cell Signaling, 2247; 1:1,500), p-Akt (Ser473) (Cell Signaling, 4060; 1:3,000), p-ERK (Thr202/Tyr204) (Cell Signaling, 4695; 1:2,000), caspase-3 (Abcam, Cambridge, UK, ab4051; 1:500), and β-actin (Proteintech, Rosemont, IL, USA, 66009-1-Ig; 1:4,000).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

doi: 10.1371/journal.pone.0067813

Figure Lengend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

Techniques: Western Blot, Labeling, Isolation

Journal: PLoS ONE

Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

doi: 10.1371/journal.pone.0067813

Figure Lengend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

doi: 10.1371/journal.pone.0067813

Figure Lengend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

doi: 10.1371/journal.pone.0067813

Figure Lengend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

Techniques: Western Blot, Labeling, Isolation

Journal: PLoS ONE

Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

doi: 10.1371/journal.pone.0067813

Figure Lengend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

doi: 10.1371/journal.pone.0067813

Figure Lengend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

Techniques: Western Blot