Journal: Breast Cancer Research : BCR

Article Title: Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

doi: 10.1186/bcr3067

Figure Lengend Snippet: HER2-overexpressing cell lines exhibit distinct responses when treated with potent anti-HER2 therapy . (A) A panel of HER2-overexpressing breast cancer cell lines was treated with lapatinib (1 μM) plus trastuzumab (10 μg/ml) for 48 h and whole-cell extracts were analyzed by immunoblotting with indicated antibodies. (B) Combination therapy (trastuzumab plus lapatinib) growth response in the HER2-overexpressing breast cancer cell line panel. Growth inhibition was determined by methylene blue assay. Shown are the percent inhibitions of cells treated for six days normalized to non-treated cells. The experiment was performed in quadruplicate. Error bars on plots represent +/- standard error (SE). (C) Growth curves of de novo resistant MDA-MB-361 cells treated with different target therapies/regimens for nine days: trastuzumab (T) (10 μg/ml), lapatinib (L) (1 μM), trastuzumab plus lapatinib (L + T), or endocrine therapy, fulvestrant (F) (10 -7 M), untreated (C). Cell numbers were quantified by absorbance at 655 nm after staining with methylene blue. Conditions were repeated in quadruplicate. Significance between groups was determined by multiple comparisons using the Sidak method (* P < 0.0001, F versus C, T, L, or L + T).

Article Snippet: Primary antibodies were: phospho-EGFR-Tyr1173 (Epitomics, Burlingame, CA, USA), EGFR, phospho-HER2- Tyr877, phospho-HER2-Tyr1221, HER2, phospho-HER3-Tyr1289, phospho-AKT-Thr308, phospho-AKT-Ser374, AKT, phospho-p44/42 MAPK- Thr202/Tyr204, p44/42 MAPK, β-actin, insulin-like growth factor-I receptor (IGF1R), cleaved PARP, caveolin-1 (Cav-1), Bik (all from Cell Signaling Technology), phospho-HER2-Tyr1248, HER3 (from Millipore, Billerica, MA, USA), ERα (Lab Vision, Fremont, CA, USA), progesterone receptor (PR), Cyclin-D1, and Bcl2 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Inhibition, Staining

Journal: Breast Cancer Research : BCR

Article Title: Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

doi: 10.1186/bcr3067

Figure Lengend Snippet: Resistant cells show greater proliferation and exhibit changes in ER and PR expression . (A) Cell proliferation assay of UACC-812 and BT474 parental and resistant (R) cells. Cells were treated with trastuzumab (T, 10 μg/ml), lapatinib (L, 1 μM), or trastuzumab plus lapatinib (L + T). After six days, viable cells were visualized by methylene blue staining and photographed. (B) Fold changes in cell growth of UACC-812 and BT474 parental and resistant cells with or without the respective anti-HER2 therapies, following six days of treatment. Cell numbers were quantified by absorbance at 655 nm and normalized against Day 0. Significance between groups was determined by multiple comparisons using the Sidak method (* P < 0.0001, P:T versus TR:T, P:L versus LR:L, or P:L + T versus LTR:L + T for both models). (C) Immunohistochemical detection of ER and PR in BT474 and UACC-812 parental and distinct resistant clones.

Article Snippet: Primary antibodies were: phospho-EGFR-Tyr1173 (Epitomics, Burlingame, CA, USA), EGFR, phospho-HER2- Tyr877, phospho-HER2-Tyr1221, HER2, phospho-HER3-Tyr1289, phospho-AKT-Thr308, phospho-AKT-Ser374, AKT, phospho-p44/42 MAPK- Thr202/Tyr204, p44/42 MAPK, β-actin, insulin-like growth factor-I receptor (IGF1R), cleaved PARP, caveolin-1 (Cav-1), Bik (all from Cell Signaling Technology), phospho-HER2-Tyr1248, HER3 (from Millipore, Billerica, MA, USA), ERα (Lab Vision, Fremont, CA, USA), progesterone receptor (PR), Cyclin-D1, and Bcl2 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Proliferation Assay, Staining, Immunohistochemical staining, Clone Assay

Journal: Breast Cancer Research : BCR

Article Title: Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

doi: 10.1186/bcr3067

Figure Lengend Snippet: BT474 lapatinib resistant cells with prolonged treatment reactivate HER receptor activity . (A) Growth curves of UACC-812 and BT474 late stage lapatinib resistant cells (LLR) treated with different targeted therapies for nine days: trastuzumab (T) (10 μg/ml), lapatinib (L) (1 μM), trastuzumab plus lapatinib, or endocrine therapy, fulvestrant (F) (10 -7 M). Significance between groups was determined by multiple comparisons using the Sidak method (* P = 0.0008, BT474 LLR + L versus LLR + L + F, * P = 0.0044, BT474 LLR + L versus LLR + L + T; * P < 0.0001, UACC-812 LLR + L versus LLR + L + F). (B) Immunohistochemical detection of ER, PR, and phospho-HER2 (Tyr877) in BT474 late stage lapatinib resistant cells. (C) Western blot analysis of UACC-812 and BT474 parental and resistant cell lines, including early (LR) and late (LLR) stage lapatinib resistant cells. Whole-cell extracts were analyzed by Western blot with the indicated antibodies. (D) BT474 parental, early, late stage lapatinib resistant, and combination (L + T) resistant cells were treated with fulvestrant for 24, 48, or 72 h and whole-cell extracts were analyzed by Western blot with the indicated antibodies.

Article Snippet: Primary antibodies were: phospho-EGFR-Tyr1173 (Epitomics, Burlingame, CA, USA), EGFR, phospho-HER2- Tyr877, phospho-HER2-Tyr1221, HER2, phospho-HER3-Tyr1289, phospho-AKT-Thr308, phospho-AKT-Ser374, AKT, phospho-p44/42 MAPK- Thr202/Tyr204, p44/42 MAPK, β-actin, insulin-like growth factor-I receptor (IGF1R), cleaved PARP, caveolin-1 (Cav-1), Bik (all from Cell Signaling Technology), phospho-HER2-Tyr1248, HER3 (from Millipore, Billerica, MA, USA), ERα (Lab Vision, Fremont, CA, USA), progesterone receptor (PR), Cyclin-D1, and Bcl2 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activity Assay, Immunohistochemical staining, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

doi: 10.1186/bcr3067

Figure Lengend Snippet: Growth of UACC-812 xenografts treated with various anti-HER2 treatments, with or without estrogen deprivation . (A) Treatment in the presence of estrogen supplementation, representing no endocrine therapy. Treatments included: Estrogen alone (E2) or with lapatinib (E2 + L), trastuzumab (E2 + T), or their combination (E2 + L + T). (B) Treatments in the presence of endocrine therapy in the form of estrogen deprivation. Treatments included: Estrogen (E2), estrogen deprivation (ED) alone, or along with lapatinib (ED + L), trastuzumab (ED + T), or their combination (ED + L + T). Results are presented as the mean tumor volume; error bars represent the standard error.

Article Snippet: Primary antibodies were: phospho-EGFR-Tyr1173 (Epitomics, Burlingame, CA, USA), EGFR, phospho-HER2- Tyr877, phospho-HER2-Tyr1221, HER2, phospho-HER3-Tyr1289, phospho-AKT-Thr308, phospho-AKT-Ser374, AKT, phospho-p44/42 MAPK- Thr202/Tyr204, p44/42 MAPK, β-actin, insulin-like growth factor-I receptor (IGF1R), cleaved PARP, caveolin-1 (Cav-1), Bik (all from Cell Signaling Technology), phospho-HER2-Tyr1248, HER3 (from Millipore, Billerica, MA, USA), ERα (Lab Vision, Fremont, CA, USA), progesterone receptor (PR), Cyclin-D1, and Bcl2 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

doi: 10.1186/bcr3067

Figure Lengend Snippet: BT474 late stage lapatinib-resistant cells overexpress HER2 and HER ligands . (A) mRNA expression levels of HER receptors and ligands in BT474 parental and distinct resistant derivatives by qRT-PCR. Data were normalized to parental cells. (B) EGFR, HER2, and HER3 protein levels in BT474 parental, early, and late stage lapatinib-resistant cells. Protein level was quantified with Odyssey software (LI-COR Biosciences, Inc., Lincoln, NE). Each expression level was acquired from three independent samples for each derivative. Significance between groups was determined by multiple comparisons using the Sidak method (* P < 0.0001, EGFR expression: P versus LLR; HER3 expression: P versus LR, P versus LLR, or LR versus LLR).

Article Snippet: Primary antibodies were: phospho-EGFR-Tyr1173 (Epitomics, Burlingame, CA, USA), EGFR, phospho-HER2- Tyr877, phospho-HER2-Tyr1221, HER2, phospho-HER3-Tyr1289, phospho-AKT-Thr308, phospho-AKT-Ser374, AKT, phospho-p44/42 MAPK- Thr202/Tyr204, p44/42 MAPK, β-actin, insulin-like growth factor-I receptor (IGF1R), cleaved PARP, caveolin-1 (Cav-1), Bik (all from Cell Signaling Technology), phospho-HER2-Tyr1248, HER3 (from Millipore, Billerica, MA, USA), ERα (Lab Vision, Fremont, CA, USA), progesterone receptor (PR), Cyclin-D1, and Bcl2 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Software

Journal: Breast Cancer Research : BCR

Article Title: Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

doi: 10.1186/bcr3067

Figure Lengend Snippet: Inhibition of HER2 restores lapatinib sensitivity in BT474 late stage lapatinib resistant cells . (A) BT474 parental and resistant cells were treated with pooled EGFR, HER2, HER3, ER siRNA, or non-targeting control siRNA, for 72 hours. Proliferation was measured using the Click-iT EdU (5-ethynyl-2'- deoxyuridine) Microplate Assay. Apoptosis was measured by detecting Annexin V expression. Signals were visualized and quantitated by the Celigo cytometer (Cyntellect, San Diego, CA, USA). (B) Down-regulation of EGFR, HER2, HER3, and ER in BT474 derivatives after siRNA treatment was detected by Western blot. Whole-cell extracts were analyzed with the indicated antibodies, including downstream signaling. (C) Growth fold change of double dosage (2 μM) lapatinib on BT474 early and late stage-lapatinib resistant cells for six-day treatment. Cell numbers were assessed using methylene blue and quantified by absorbance at 655 nm and normalized against Day 0. Significance between groups was determined by multiple comparisons using the Sidak method (* P < 0.0001, LLR + L versus LLR + 2L).

Article Snippet: Primary antibodies were: phospho-EGFR-Tyr1173 (Epitomics, Burlingame, CA, USA), EGFR, phospho-HER2- Tyr877, phospho-HER2-Tyr1221, HER2, phospho-HER3-Tyr1289, phospho-AKT-Thr308, phospho-AKT-Ser374, AKT, phospho-p44/42 MAPK- Thr202/Tyr204, p44/42 MAPK, β-actin, insulin-like growth factor-I receptor (IGF1R), cleaved PARP, caveolin-1 (Cav-1), Bik (all from Cell Signaling Technology), phospho-HER2-Tyr1248, HER3 (from Millipore, Billerica, MA, USA), ERα (Lab Vision, Fremont, CA, USA), progesterone receptor (PR), Cyclin-D1, and Bcl2 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Inhibition, Expressing, Cytometry, Western Blot

Journal: PLoS ONE

Article Title: SPIN90 Knockdown Attenuates the Formation and Movement of Endosomal Vesicles in the Early Stages of Epidermal Growth Factor Receptor Endocytosis

doi: 10.1371/journal.pone.0082610

Figure Lengend Snippet: A. (a) Control and SPIN90 knockdown (KD) HeLa cells were starved for 16 h, and treated with 40 ng/ml EGF for the indicated times. One set of SPIN90 knockdown cells was treated with 40 ng/ml EGF and 50 µM PD98059 for the indicated times (KD+PD98059). Cell lysates were immunoblotted with the indicated antibodies. (b) Band intensities was normalized to that of tubulin. Bar graphs represent EGFR stability, calculated as the ratio of band intensity of EGFR at the indicated time-points (Ii), compared to that at 0 time (Io). (c) ERK activity is presented as the ratio of band intensity of phospho-ERK (pERK), compared to that of ERK. (d) Experiments were performed the same as Fig. 6A. Cell lysates were immunoblotted with the indicated antibodies. (e) Bar graphs represent active Akt, calculated as the ratio of band intensity of phosphor-Akt (pAkt) compared to total Akt.. B. Control, SPIN90 knockdown (KD) and PD98059 (50 µM)-treated SPIN90 KD HeLa cells starved for 16 h were incubated with 40 ng/ml EGF for the indicated times. PD98059 (50 µM) was used. Cyclin D1 was detected via immunoblot analysis, and the relative values of band intensities calculated. C . Control, SPIN90 knockdown (KD) and PD98059 (50 µM)-treated SPIN90 KD HeLa cells were incubated with 40 ng/ml EGF or 5% FBS for 36 h, and the MTT assay performed as described in Materials & Methods. Lysates dissolved with DMSO were analyzed by monitoring absorbance at 560 and 670 nm. Four independent replicates were performed for each experiment ( p value; * < 0.01, ** < 0.005, *** < 0.001).

Article Snippet: Mouse monoclonal antibodies used against phospho-ERK and ERK were obtained from Cell Signaling (Denver, USA), and anti-EGFR (528) monoclonal antibody from Santa Cruz Biotechnology.

Techniques: Activity Assay, Incubation, Western Blot, MTT Assay

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: 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: International Journal of Nanomedicine

Article Title: Investigation of antitumor activities of trastuzumab delivered by PLGA nanoparticles

doi: 10.2147/IJN.S152742

Figure Lengend Snippet: HER2 binding of released TZ from PLGA NPs. Notes: ( A ) TZ-FITC binding on SKBR3 cells after treatment with RIgG, CTZ and RTZ at two different concentrations (2 and 20 µg mL −1 ) for 24 and 48 h. Data are represented as the mean of fluorescence intensity of three independent replicates ± SD acquiring 10,000 events for each sample by flow cytometry. ( B – E ) Confocal microscopy images of TZ-FITC binding on SKBR3. ( B ) Basal binding of TZ-FITC; ( C , D and E ) binding of TZ-FITC after pre-treatment of RIgG, CTZ and RTZ, respectively, for 24 h at 37°C. Cells were labeled with DAPI (nuclei) and WGA-555 (membranes). Scale bar =50 µm. **** P <0.01 vs UNTR; ** P <0.05 vs UNTR. Abbreviations: HER2, human epidermal growth factor receptor 2; TZ, trastuzumab; PLGA NPs, poly(lactic- co -glycolic) acid nanoparticles; FITC, fluorescein isothiocyanate; RIgG, released IgG; CTZ, control TZ; RTZ, released TZ; SD, standard deviation; CNTR, cells without any labeling; UNTR, cells coated with TZ-FITC.

Article Snippet: The membranes were blocked with TBS – Tween 0.1% solution +5% milk and then immunodecorated alternatively with monoclonal anti-HER2/ErbB2 antibody (Cell Signaling Technology, Leiden, the Netherlands) or polyclonal anti-phosphorylated HER2 antibody (Tyr1248; Cell Signaling Technology) or monoclonal antibody anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA) in TBS – Tween 0.1% solution +5% BSA overnight at 4°C.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Confocal Microscopy, Labeling, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Investigation of antitumor activities of trastuzumab delivered by PLGA nanoparticles

doi: 10.2147/IJN.S152742

Figure Lengend Snippet: HER2 phosphorylation. Notes: ( A ) Analysis of pHER2 (Y1248) and HER2 expression on SKBR3 cells after treatment with control CTZ and RTZ at 2 µg mL −1 for 4 and 24 h; ( B ) Analysis of HER2 expression on SKBR3 cells after treatment with PLGA-TZ at 2 µg mL −1 for 4 and 24 h. Values were calculated as ratio between pHER2/HER2 and HER2/b-actin and normalized with untreated cells (UNTR). Abbreviations: HER2, human epidermal growth factor receptor 2; CTZ, control trastuzumab; RTZ, released trastuzumab; PLGA-TZ, trastuzumab-loaded poly(lactic- co -glycolic) acid nanoparticles; UNTR, cells without treatment.

Article Snippet: The membranes were blocked with TBS – Tween 0.1% solution +5% milk and then immunodecorated alternatively with monoclonal anti-HER2/ErbB2 antibody (Cell Signaling Technology, Leiden, the Netherlands) or polyclonal anti-phosphorylated HER2 antibody (Tyr1248; Cell Signaling Technology) or monoclonal antibody anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA) in TBS – Tween 0.1% solution +5% BSA overnight at 4°C.

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