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: Cell reports

Article Title: KRAS G12C -independent feedback activation of wild-type RAS constrains KRAS G12C inhibitor efficacy

doi: 10.1016/j.celrep.2022.110993

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal Anti-phospho-HER2 Y1248 , Cell Signaling Technologies , Cat# 2247S.

Techniques: Derivative Assay, Recombinant, In Vitro, In Vivo, Software

Journal: PLoS ONE

Article Title: The Marine-Derived Oligosaccharide Sulfate (MdOS), a Novel Multiple Tyrosine Kinase Inhibitor, Combats Tumor Angiogenesis both In Vitro and In Vivo

doi: 10.1371/journal.pone.0003774

Figure Lengend Snippet: A–B, MdOS blocks HER-2 phosphorylation and its downstream signal transduction in SK-OV-3 cells (A) and NIH-3T3/neu cells (B). C–D, Inhibition of EGF-stimulated EGFR phosphorylation and signal transduction by MdOS in A431 (C) and MDA-MB-468 (D) cells. Serum-starved cells were incubated with indicated concentrations of MdOS for 6 h at 37°C, EGF (50 ng/ml) was added to the cultures during the last 15-min treatment. E–F, Inhibition of VEGF-stimulated VEGFR2 phosphorylation and signal transduction by MdOS. HMEC (E) and NIH-3T3/flk-1 (F) cells were starved, then incubated with indicated concentrations of MdOS for 6 h at 37°C, VEGF (50 ng/ml) was added to the cultures during the last 15-min treatment. Protein samples were separated by SDS-PAGE and probed using the indicated antibodies. Representative data are shown.

Article Snippet: Western blot analyses were subsequently performed as previous described , with the antibodies against phospho-EGFR (Tyr1068) (#2234), phospho-HER-2 (Tyr1248) (#2247), phospho-VEGFR2 (Tyr996) (#2474), VEGFR2 (#2472), phospho-ERK1/2 (#9101), ERK1/2 (#9102), phosphor-AKT (Ser473) (#9271) and AKT (#9272) (Cell Signaling Technology, Beverly, MA, USA), EGFR (sc-03), HER-2(sc-284) and actin (sc-8432) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transduction, Inhibition, Incubation, SDS Page

Journal: PLoS ONE

Article Title: The Marine-Derived Oligosaccharide Sulfate (MdOS), a Novel Multiple Tyrosine Kinase Inhibitor, Combats Tumor Angiogenesis both In Vitro and In Vivo

doi: 10.1371/journal.pone.0003774

Figure Lengend Snippet: VEGFR2 was injected at the concentrations of 2.56, 0.64, 0.32, 0.16 and 0.08 µM (from top to bottom); HER-2 was injected at the concentrations of 0.53, 0.26, 0.13, 0.07 and 0.03 µM (from top to bottom); EGFR was injected at the concentrations of 6, 3, 1.5, 0.75, 0.38, 0.19 and 0.09 µM (from top to bottom); 6×his-tag was injected at the concentrations of 100, 10, 1, 0.1, 0.01 µM. Sensorgram responses at equilibrium were plotted against each concentration of compounds and the equilibrium dissociation constant (K D ) of the binding system was calculated using BIAeval software 3.1.

Article Snippet: Western blot analyses were subsequently performed as previous described , with the antibodies against phospho-EGFR (Tyr1068) (#2234), phospho-HER-2 (Tyr1248) (#2247), phospho-VEGFR2 (Tyr996) (#2474), VEGFR2 (#2472), phospho-ERK1/2 (#9101), ERK1/2 (#9102), phosphor-AKT (Ser473) (#9271) and AKT (#9272) (Cell Signaling Technology, Beverly, MA, USA), EGFR (sc-03), HER-2(sc-284) and actin (sc-8432) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Injection, Concentration Assay, Binding Assay, Software

Journal: PLoS ONE

Article Title: The Marine-Derived Oligosaccharide Sulfate (MdOS), a Novel Multiple Tyrosine Kinase Inhibitor, Combats Tumor Angiogenesis both In Vitro and In Vivo

doi: 10.1371/journal.pone.0003774

Figure Lengend Snippet: VEGFR2 (A) and HER-2 (B) and EGFR (C) kinase assays were performed as described in in the presence of varying concentrations of ATP. Initial reaction velocity was expressed as the phosphorylation of poly(Glu, Tyr) 4∶1 substrate. All x, y data sets were multiplied by 100 for purposes of graphical presentation.

Article Snippet: Western blot analyses were subsequently performed as previous described , with the antibodies against phospho-EGFR (Tyr1068) (#2234), phospho-HER-2 (Tyr1248) (#2247), phospho-VEGFR2 (Tyr996) (#2474), VEGFR2 (#2472), phospho-ERK1/2 (#9101), ERK1/2 (#9102), phosphor-AKT (Ser473) (#9271) and AKT (#9272) (Cell Signaling Technology, Beverly, MA, USA), EGFR (sc-03), HER-2(sc-284) and actin (sc-8432) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques:

Journal: OncoTargets and therapy

Article Title: Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2

doi: 10.2147/OTT.S82225

Figure Lengend Snippet: SHP-1 binds to EGFR and HER-2 protein. Notes: ( A , B ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. EGFR and HER-2 proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( C , D ) SKBR3 cells were transfected with control (NC), SHP-1 WT, or SHP-1 MT vector for 48 hours. Phosphorylated-EGFR (pEGFR) and phosphorylated-HER-2 (pHER-2) proteins were pulled down by immunoprecipitation and the protein level was detected by immunoblotting. ( E , F ) The binding between SHP-1 and HER-3 or HER-4 was also evaluated through immunoprecipitation. The whole gel is shown, and no specific band of HER-3 or HER-4 was detected. ( G ) Bimolecular fluorescence complementation (BiFC) was performed to confirm the binding activity between SHP-1 and EGFR, HER-2, HER-3, and HER-4. BiFC signals were generated by interaction of the GFP fluorophore components based on proximity. + and − indicate receiving and withholding of the treatment, respectively. Abbreviations: EGFR, epidermal growth factor receptor; HER-2, human epidermal receptor; NC, nonsense control; WT, wild-type; IP, immunoprecipitation.

Article Snippet: All antibodies were purchased from Cell Signaling Technologies: phospho-HER2 (Tyr1248) rabbit polyclonal antibody (#2247), HER2 rabbit polyclonal antibody (#2242), phospho-EGFR (Tyr1173) rabbit monoclonal antibody (#4407), EGFR rabbit monoclonal antibody (#4405), phospho-Erk1/2 (Thr202/Tyr204) rabbit monoclonal antibody (#4370), Erk1/2 rabbit monoclonal antibody (#4695), phospho-Stat3 (Tyr705) rabbit monoclonal antibody (#9145), Stat3 rabbit monoclonal antibody (#4904), phospho-Akt (Ser473) rabbit monoclonal antibody (#4060), and Akt (pan) rabbit monoclonal antibody (#4685).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Fluorescence, Activity Assay, Generated

Journal: Cancers

Article Title: HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer

doi: 10.3390/cancers14164054

Figure Lengend Snippet: IgG purified from ECTM- and Δ16ECTM-immune sera impaired ERK activation in BT-474 BC cells. ( A ). Representative Western blot analysis of HER2 downstream signaling pathways in BT-474 cells, treated or not with trastuzumab or control-IgG or immune-IgG at the indicated concentrations for 8 h. Expression level of HER2, pHER2, AKT, pAKT, ERK, pERK was analyzed. Equal amounts of protein (20 μg) were loaded and β-actin was used as loading control. Data are representative of a typical experiment repeated three times with similar results. ( B ). Densitometric quantification of HER2 and ERK expression, normalized on β-actin, and of pERK/ERK from three independent experiments were shown; one-way ANOVA test followed by Tukey’s multiple comparison test (* p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001: IgG ECTM or IgG Δ16ECTM vs. IgG empty; trastuzumab vs. control). ( C ). IgG purified from ECTM- and Δ16ECTM-immune sera reduced the levels of phosphorylated RB in BT-474 cells. Upper panel: representative Western blot analysis of phosphorylated RB (Ser 780) in BT-474 cells, treated or not with 15 μg/mL trastuzumab or control-IgG or immune-IgG (30 μg/mL) for 8 h. Equal amounts of protein (20 μg) were loaded and vinculin was used as a loading control. Data are representative of a typical experiment repeated three times with similar results. Lower panel: densitometric quantification of phosphorylated RB expression, normalized on vinculin, was shown; one-way ANOVA test followed by Tukey’s multiple comparison test **** p ≤ 0.0001: IgG ECTM or IgG Δ16ECTM vs. IgG empty; trastuzumab vs. control. The original western blot data can be found in .

Article Snippet: Primary antibodies to HER2 (#4290, lot 2), pHER2 (#2247, lot 9), ERK (#4695, lot 14), pERK (#4370, lot 12), Akt (#9272, lot # 24), pAkt (#9271, lot # 13), β-actin (#2247, lot 9) were from Cell Signaling Technology (Danvers, MA, USA) (1:1000).

Techniques: Purification, Activation Assay, Western Blot, Expressing

Journal: Cancers

Article Title: HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer

doi: 10.3390/cancers14164054

Figure Lengend Snippet: IgG purified from ECTM- and Δ16ECTM-immune sera impaired ERK activation in trastuzumab-resistant BT-474 BC cells. ( A ). Representative Western blot analysis of HER2 downstream signaling pathways in trastuzumab-resistant BT-474 cells, treated or not with trastuzumab or control-IgG or immune-IgG at the indicated concentrations for 8 h. Expression level of HER2, pHER2, ERK, pERK was analyzed. Equal amounts of protein (20 μg) were loaded and β-actin was used as loading control. Data are representative of a typical experiment repeated three times with similar results. ( B ). Densitometric quantification of HER2 and ERK expression, normalized on β-actin, and of pERK/ERK from three independent experiments were shown; one-way ANOVA test followed by Tukey’s multiple comparison test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001: IgG ECTM or IgG Δ16ECTM vs. IgG empty/control). ( C ). IgG purified from ECTM- and Δ16ECTM-immune sera reduced the levels of phosphorylated RB in trastuzumab-resistant BT-474.R BC cells. Upper panel: representative Western blot analysis of phosphorylated RB (Ser 780) in trastuzumab-resistant BT-474.R BC cells, treated or not with 15 μg/mL trastuzumab or control-IgG or immune-IgG (30 μg/mL) for 8 h. Equal amounts of protein (20 μg) were loaded and tubulin was used as loading control. Data are representative of a typical experiment repeated three times with similar results. Lower panel: densitometric quantification of phosphorylated RB expression, normalized on tubulin, was shown; one-way ANOVA test followed by Tukey’s multiple comparison test **** p ≤ 0.0001: IgG ECTM or IgG Δ16ECTM vs. IgG empty. The original western blot data can be found in (pages 4–6).

Article Snippet: Primary antibodies to HER2 (#4290, lot 2), pHER2 (#2247, lot 9), ERK (#4695, lot 14), pERK (#4370, lot 12), Akt (#9272, lot # 24), pAkt (#9271, lot # 13), β-actin (#2247, lot 9) were from Cell Signaling Technology (Danvers, MA, USA) (1:1000).

Techniques: Purification, Activation Assay, Western Blot, Expressing