Antibodies against TDP‐43 (Sigma‐Aldrich Cat# SAB4200006, Cosmo Bio Co Cat# CAC‐TIP‐TD‐P09), GFP (UC Davis/NIH NeuroMab Facility Cat# 73‐132), β‐actin (Sigma‐Aldrich Cat# A5316), calnexin (Enzo Life Sciences Cat# ADI‐SPA‐860‐F), VPS4B (Sigma‐Aldrich Cat# PA5‐30316 for IHC and Proteintech Group Cat# 17673‐1‐AP for immunoblotting and immunofluorescence), ErbB4 (Santa Cruz Biotechnology Cat# sc‐283), phosphor‐ErbB4‐Y1056 (Santa Cruz Biotechnology Cat# sc‐33040), FGFR1 (Novus Cat# NB600‐1287), phosphor‐FGFR1‐Y654 (Abcam Cat# ab59194), myc‐tag (Santa Cruz Biotechnology Cat# sc‐40), HA‐tag (Sigma‐Aldrich Cat# H9658 and Roche Cat# 3F10), anti‐mouse and rabbit HRP‐coupled secondary antibodies and anti‐mouse and rabbit Alexa‐coupled secondary antibodies (Life Technologies) are commercially available. TO‐PRO 3 and transferrin‐Alexa 555 were purchased from Life Technologies, and DAPI and EZ‐Link sulfo‐NHS‐LC biotin from Sigma‐Aldrich. β2‐Transferrin protein levels from undiluted CSF samples derived from sporadic ALS patients and controls were quantified using a commercially available ELISA kit (www.mybiosource.com , MBS923983) following the manufacturer's instructions. Recombinant NRG1 and FGF1 were purchased from R&D Systems.
Erbb4
Manufactured by Santa Cruz Biotechnology
Sourced in United States
ErbB4 is a transmembrane protein that functions as a receptor tyrosine kinase. It is a member of the ErbB family of receptors and plays a role in signal transduction pathways.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Calnexin, by Enzo Life Sciences (1 mentions)
Ha tag, by Merck Group (1 mentions)
Myc tag, by Santa Cruz Biotechnology (1 mentions)
Vps4b, by Merck Group (1 mentions)
Fgfr1, by Novus Biologicals (1 mentions)
Ez link sulfo nhs lc biotin, by Merck Group (1 mentions)
Recombinant nrg1, by R&D Systems (1 mentions)
To pro 3, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Trypan blue solution 0.4, by Merck Group (1 mentions)
Cxcl5, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Trans blot turbo transfer, by Bio-Rad (1 mentions)
Mini protean tetra cell unit, by Bio-Rad (1 mentions)
7 protocols using erbb4
1
Immunological Analysis of ALS Biomarkers
2
Cell Signaling Pathway Analysis
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Recombinant human (rh) CXCL1, CXCL5, CXCL6, M-CSF and RANKL were purchased from PeproTech and rh NDP from R&D Systems. Primary antibodies were produced in-house (ErbB4) or purchased from Santa Cruz Biotechnology (p-Tyr, NRG3). Trypan Blue solution 0.4% was delivered by Sigma-Aldrich, cell culture media and supplements by Gibco (Life Technologies), and epidermal growth factor (EGF) and Matrigel from BD Biosciences.
3
Western Blot Analysis of Cellular Signaling
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Protein samples were diluted with Laemmli buffer, separated on Criterion Stain-Free precast gels in a BioRad Mini PROTEAN Tetra-Cell unit and transferred to nitrocellulose membranes using a BioRad Trans Blot Turbo transfer. Membranes were blocked with 5% nonfat dry milk in Tris buffered saline (pH 7.5) containing 0.1% Tween 20 (TBST) at room temperature for 1h. Then, membranes were incubated in 2% BSA with the relevant primary antibodies at 4°C overnight. Anti-AKT (1/1000), -p-AKT Ser473 (1/1000), -FOXO (1/1000), -p-FOXO, (1/1000), -ERBB3 (1/200) and–p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA). Anti-ERBB-2 (1/200), -ERBB-4 (1/200), -p-ERBB-2 (1/200), -p-ERBB4 (1/200) and -NRG1 N120A/9 antibodies were purchased from Santa Cruz (Santa Cruz Biotechnology, CA). After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody in TBST at room temperature for 1 hour, enhanced chemiluminescence (ECL) reagents (Pierce) were used to detect interactions and digital images were acquired using the Molecular Imager ChemiDoc XRS System (Biorad). Signals were quantified using the Image Lab 4.1 software (BioRad) and normalized using the Total Protein Normalization (TPN) method provided by Stain Free technology.
4
Profiling EGFR Signaling Pathways in Lung Cancer Cells
5
Immunohistochemical Profiling of ERBB Receptors
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Expression of ERBB1-4 receptor proteins was determined by IHC using commercial monoclonal antibodies EGFR (1:50, Santa Cruz), ERBB-2 (1:50, Santa Cruz), ERBB-3 (1:50, Santa Cruz) and ERBB-4 (1:50, Santa Cruz). The proliferation of tumor cells was detected by ki-67 IHC staining. Formalin-fixed and paraffin-embedded sections, 4 μm thick, with representative tumor tissue, were incubated with primary antibodies after antigen retrieval by pressure autoclaving. An automatized histostainer was used for the immunohistochemcial procedures (Dako Autostainer, Denmark). For visualization of immunoreactivity, DAKO EnVision system was used with diaminobenzidin as chromogene. Sections were counterstained with haematoxylin. Positive controls were included in each staining run.
The immunoreactivity was assessed by means of intensity and percentage of immunoreactive tumor cells. Intensity was recorded as 0 (no reaction) to 3 (strong reaction). Fraction of immunoreactive tumor cells was recorded as 0 (no positive cells), 1 (<10% positive cells), 2 (10-50% positive cells), or 3 (>50% positive cells). A staining index was calculated as the product of intensity and fraction of positive tumor cells [15 ].
The immunoreactivity was assessed by means of intensity and percentage of immunoreactive tumor cells. Intensity was recorded as 0 (no reaction) to 3 (strong reaction). Fraction of immunoreactive tumor cells was recorded as 0 (no positive cells), 1 (<10% positive cells), 2 (10-50% positive cells), or 3 (>50% positive cells). A staining index was calculated as the product of intensity and fraction of positive tumor cells [15 ].
6
Western Blot Analysis of Neuronal Markers
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Brain tissues were homogenized in the sample lysis buffer (62.5 mM Tris-HCl pH 6.8, 2 % SDS, 0.5 % NP-40, 5 mM EDTA) plus the protease inhibitor cocktail (Roche). Protein samples (15-30 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Memb-ranes were probed with antibodies directed against phosphotyrosine (1:1000, Millipore, Bedford, MA, USA), ErbB4 (1:1000, Santa Cruz Biotechnology). Alter-natively, immunoblots were probed with antibodies directed against excitatory and inhibitory neuronal markers GluA1 (1:500, Millipore), GluA2/3 (1:1000, Millipore), GluA4 (1:1000, Millipore), GluN1 (1:1000, Millipore), GluN2A (1:500, Millipore), GluN2B (1: 500, Millipore), GluN2C (1:500, Millipore), GluN2D (1:1000, Santa Cruz Biotechnology), GAD65/67 (1:1000, Millipore), parvalbumin (1:3000, Abcam, Cambridge UK), tyrosine hydroxylase (1:1000, Millipore) neuregulin-1 (1:300, RandD Systems, Minneapolis, MN), and β-actin (1:2000, Millipore). Immunoreactivity on membranes was detected by peroxidase-conjugated anti-immunoglobulin antibodies followed by chemiluminescence reaction (ECL kit, GE Healthcare) and film exposure. The intensity of an immunoreactive band, whose size matched the authentic molecular weight, was measured by an image processing software, GENETOOLS (Syngene, Cambridge, UK).
7
Immunohistochemical Analysis of Tumor Samples
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Five of 16 tumor samples, for which paired adjacent normal tissues were available, were used to perform WES. Five FFPE paired tumor and adjacent normal tissue blocks were cut into 4μm sections, which were deparaffinized in xylene and hydrated by immersion in a graded ethanol series. Antigen retrieval was performed in a microwave by placing sections in epitope retrieval solution (0.01 M citrate buffer, pH 6.0, or 10 mM ethylenediaminetetraacetic acid, pH 8.0) for 20 min, and endogenous peroxidase was blocked via immersion in 0.3% hydrogen peroxide for 10 min. IHC staining was performed using the Dako Auto stainer plus Universal Staining System (Dako Cytomation, Carpinteria, CA, USA) with a Chem Mate DAKO En Vision detection kit (Dako Cytomation). Antibodies against chromogranin A (DAKO, Glostrup, Denmark), synaptophysin (Ventana, Roche, Tucson, USA), Ki-67 (monoclonal, MIB-1 clone, 1:50, DAKO), and ERBB4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.
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