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: Neuro-oncology Advances

Article Title: EGFR blockade in GBM brain tumor stem cells synergizes with JAK2/STAT3 pathway inhibition to abrogate compensatory mechanisms in vitro and in vivo

doi: 10.1093/noajnl/vdaa020

Figure Lengend Snippet: Afatinib effectively decreases BTSC viability and sphere-forming capacity and has on-target activity on phospho-EGFR. (A) Afatinib decreased cell viability in molecularly diverse BTSCs. Normal human astrocytes were unaffected. (B) Representative images of BT73 ( EGFRvIII mt) spheres following afatinib treatment. The scale bar represents 300 μm. (C) Quantification for 2 representative BTSC cultures, BT50 ( EGFR wt) and BT73 ( EGFRvIII mt) (* P < .05, ** P < .01, *** P < .001, and **** P < .0001 vs DMSO; Sidak’s multiple comparison two-way ANOVA). Error bars represent SD. (D) Afatinib demonstrates on-target activity as seen by a decrease in p-EGFR Y1068 levels. An increase in p-STAT3 Y705 levels was observed (representative BTSC cultures, BT50 [ EGFR wt] and BT73 [ EGFRvIII mt] shown).

Article Snippet: Primary antibodies included p-STAT3 Y705 (1:1000; Cell Signaling Technology [CST]), STAT3 (1:1000; CST), p-EGFR Y1068 (1:1000; CST), EGFR (1:1000; CST), p-p44/42 MAPK (T202/Y204) (1:1000; CST), p44/42 MAPK (1:4000; CST), β-tubulin (1:1000; CST), and Actin (1:1000; Santa Cruz Biotechnology).

Techniques: Activity Assay

Journal: Neuro-oncology Advances

Article Title: EGFR blockade in GBM brain tumor stem cells synergizes with JAK2/STAT3 pathway inhibition to abrogate compensatory mechanisms in vitro and in vivo

doi: 10.1093/noajnl/vdaa020

Figure Lengend Snippet: Afatinib and pacritinib effectively penetrate the brain and demonstrate on-target activity in orthotopic BTSC xenografts. (A) Brain concentrations of afatinib and pacritinib at 300 min post-dosing with single agents or concurrent administration. Values represent mean ± SD (** P < .0014; unpaired t -test). (B) Anti-human nucleolin was used to confirm the presence of tumor burden. (C) A decrease in positive p-EGFR Y1068 staining was seen following 15 mg/kg treatment of afatinib. There was a further decrease in positive p-EGFR Y1068 staining for the combination-treated group. (D) An increase in positive p-STAT3 Y705 signal was seen in the 15 mg/kg afatinib treated group. The increased activation of STAT3 was abolished in the combination group. The scale bar represents 100 μm and applies to all of the higher magnification images.

Article Snippet: Primary antibodies included p-STAT3 Y705 (1:1000; Cell Signaling Technology [CST]), STAT3 (1:1000; CST), p-EGFR Y1068 (1:1000; CST), EGFR (1:1000; CST), p-p44/42 MAPK (T202/Y204) (1:1000; CST), p44/42 MAPK (1:4000; CST), β-tubulin (1:1000; CST), and Actin (1:1000; Santa Cruz Biotechnology).

Techniques: Activity Assay, Staining, Activation Assay