Journal: PLoS ONE

Article Title: Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies

doi: 10.1371/journal.pone.0112376

Figure Lengend Snippet: A) Serum-starved BT-474, NCI-N87 and ACHN cells were treated with appropriate antibodies or PBS for 30 minutes followed by HRGβ stimulation for 10 minutes. B) NCI-N87 cells were treated with either the A5/F4 oligoclonal or PBS and stimulated with HRGβ, as appropriate, over a 60 minute time course. Cell lysates were analyzed by western blot and probed with anti-pAKT(S473) as a marker for ligand-induced signaling. Membranes were probed with anti-beta actin mAb that served as loading control.

Article Snippet: After overnight serum starvation in 0.1–0.5% serum containing media, cells were pretreated with appropriate antibodies or PBS for 30 minutes and stimulated with 20 nM HRG-beta (R&D systems #377-HB-050) for indicated times.

Techniques: Western Blot, Marker, Control

Journal: Nature neuroscience

Article Title: A glycolytic shift in Schwann cells supports injured axons

doi: 10.1038/s41593-020-0689-4

Figure Lengend Snippet: a, Top: Scheme of metabolomic analysis using extracts from nerve segments. Bottom: Concentrations of key energy metabolism intermediates in control and axotomized nerve segments from C57Bl/6J mice (F6P/G6P: fructose-6-phosphate/glucose-6-phosphate. FBP: fructose-1,6-bisphosphate. GI-OH3P: Glyceraldehyde-3-phosphate. 2PG/3PG: 2-phosphoglycerate/3-phosphoglycerate. LACT: lactate) (Error bars represent s.e.m. n=5 mice per condition and metabolite). b, Top: Scheme of extracellular flux analysis of SCs purified from C57Bl/6J mouse nerves. Bottom: Box and whiskers plot (maximum, 25th percentile, median, 75th percentile, minimum) shows glycolytic activity parameters as assessed by extracellular acidification rate (ECAR) measurements in control mouse SCs and cells with Nrg1-induced ErbB2 activation (n=9 well preparations per condition, *P=0.0080, **P=0.0464, ***P<0.0001). c, Western blot analysis (cropped blot images) of control- and Nrg1-treated C57Bl/6J mouse SCs probed with the indicated antibodies. (n=6 independent pair preparations (2 separate dishes) for PFKFB3, and n=3 independent pair preparations (2 separate dishes) for LDHA quantification). d, Intracellular and extracellular (supernatant) lactate concentrations from control and Nrg1-treated mouse SC preparations normalized to cell number and cellular protein. Note decreased intracellular and increased extracellular lactate levels in SCs treated with Nrg1 for 24h, indicating greatly increased lactate extrusion (Error bars represent s.e.m. n=3 well preparations from 3 independent experiments per condition).

Article Snippet: SCs were subsequently control-treated or treated with 200 ng/ml recombinant Nrg1 (R&D Systems, 396-HB-050) for 24h, collected in RIPA buffer containing phosphatase and protease inhibitors, and then processed for protein analysis and western blotting using standard procedures.

Techniques: Control, Purification, Activity Assay, Activation Assay, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers

doi: 10.1186/s13058-018-1023-x

Figure Lengend Snippet: Human epidermal growth factor receptor 3 ( HER3)-specific antibody responses are induced by adenovirus encoding full length human HER3 (Ad-HER3) in vivo. a Binding of vaccine-induced antibody (VIA) in serum from mice immunized with Ad-HER3 (HER3-VIA), Ad-lacZ (lacZ-VIA), and Ad-green fluorescence protein (GFP-VIA). Serum at dilutions presented were mixed with the HER3-4 T1 cell line or wild-type 4 T1 and binding of antibody was identified with near infrared (nIR) dye-conjugated anti-mouse IgG and detected by a LI-COR Odyssey Imager. The difference in fluorescence intensity between HER3-4 T1 and 4 T1 is graphed. b Binding of HER3-VIA to breast cancer cell lines: a panel of human breast cancer cell lines were incubated with dilutions of the HER3-VIA, washed, and then mixed with a phycoerythrin (PE)-conjugated secondary antibody. HER3-VIA binding was analyzed based on fluorescence activated cell sorting analysis and mean fluorescence intensity was reported. c Flow cytometric analysis was used to identify percentage of BT474 cells able to bind HER3-VIA (1:100 dilution). d Binding of His-tagged heregulin to HER3 receptor was measured by pre-incubating BT474 cells with heregulin (0, 10, 100 nM), or HER3-VIA (1:100) for 10 min on ice, followed by incubation with His-tagged heregulin (100 nM) for 10 min. Receptor-bound His-tagged heregulin was visualized by staining with PE-conjugated anti-His Tag antibody. Mean fluorescence intensity is shown in each histogram

Article Snippet: Heregulin (377-HB/CF), heregulin with a C-terminal 6-His tag (5898-NR) and allophycocyanin (APC)-conjugated anti-His Tag antibody (IC050A) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: In Vivo, Binding Assay, Fluorescence, Incubation, FACS, Staining