recombinant human erbb4 chimeric protein  (R&D Systems)

Journal: Cell Death Discovery

Article Title: NRG1 and KITL signal downstream of retinoic acid in the germline to support soma-free syncytial growth of differentiating spermatogonia

doi: 10.1038/cddiscovery.2015.18

Figure Lengend Snippet: ERBB-family signaling molecules in rat testis cells. ( a ) Polypeptides in the EGF super-family signal by activating ERBB-family transmembrane receptor tyrosine kinases. ERBB1 is a receptor for ‘classical’ low molecular weight EGF-like peptides. ERBB2 is the primary transducer for ligand-bound ERBB1, ERBB3 and ERBB4. ERBB2’s extracellular domain does not bind known ligands. ERBB3 is a receptor for Neuregulin-1 (NRG1), NRG2 and Neuroglycan-C (CSPG5). Ligand bound ERBB3 displays poor kinase activity and signals most effectively as a heteromer with ERBB1, ERBB2 and/or ERBB4. ERBB4 is a receptor for NRG1, NRG2, NRG3 and NRG4 plus other EGF-like peptides*. ( b ) Western blotting analysis of ERBB-family proteins in fractions of testis cells from 23-day-old rats. Lysates of type A spermatogonia after proliferating for ~180 days/15 passages in culture (SgL), freshly isolated laminin-binding type A spermatogonia (Sg), laminin non-binding spermatogenic cells (Scy), tubular somatic cells (SC), interstitial somatic cells (IC), MCF7 human mammary gland cells (MCF) and COS7 monkey kidney cells (COS). Arrowheads: ERBBs 1–4 (~185 kDa), RET (~155 and 170 kDa) and TUBA1a (~55 kDa). ( c ) Relative abundance (qtPCR) of ERBB-family transcripts in testis cells isolated from 23-day-old rats ( n =cells from three different rats; ±S.E.M.). Spermatogonia (Sg), Spermatocytes (Scy; differentiating spermatogonia/early spermatocytes), Tubular somatic cells (SC) and Interstitial somatic cells (IC) are cell types described in panel ( b ). ( d ) Testis cross-section from 26-day-old tg GCS-EGFP transgenic rats labeled with anti-ERBB2 (Red) overlaying EGFP fluorescence from germ cells (green). Note, cytoplasmic ERBB2 labeling in germ cells resembling differentiating spermatogonia (white arrows) and spermatocytes (yellow arrow). Scale, 40 μ m. ( e ) Rat seminiferous tubule whole mount from 24-day-old wild-type rat labeled using antibodies to ERBB2 (Red) and ZBTB16 (Green). Scale, 20 μ m. Note: nuclear ZBTB16 labeling is more robust in ERBB2-dim spermatogonia (cyan arrows), compared with ERBB2-bright spermatogenic cells (white arrows). ( f ) Rat seminiferous tubule whole mount from a 24-day-old wild-type rat labeled with antibodies to ERBB2 (Red) and phospho-Histone-3 (pH3, Green). Scale, 40 μ m. Note: nuclear pH3 in large mitotic ERBB2 + syncytia.

Article Snippet: Lapatinib (L-4804) was from LC Laboratories (Woburn, MA, USA); ERBB2-neutralizing IgG (AF1129), ERBB3 (348-RB) and ERBB4 (1131-ER) Fc chimeric proteins were from R&D Systems.

Techniques: Molecular Weight, Activity Assay, Western Blot, Isolation, Binding Assay, Transgenic Assay, Labeling, Fluorescence

Journal: Cell Death Discovery

Article Title: NRG1 and KITL signal downstream of retinoic acid in the germline to support soma-free syncytial growth of differentiating spermatogonia

doi: 10.1038/cddiscovery.2015.18

Figure Lengend Snippet: Spermatogenic cells selectively express neuregulin-family genes. ( a ) Strategy to analyze Neuregulin-1 ( Nrg1 ) mRNA splice variants. Exons (boxes) in Types I, II and III Nrg1 . Distinct NRG1 Types are generated by alternatively splicing N-terminal exons (NDF, Kringle and CRD domains). IgG and Sp exons encode heavily glycosylated domains, which bind heparin sulfate proteoglycans. EGF homology domains (black boxes) together with either α or β domains (gray boxes) bind with high affinity to ERBB3 and ERBB4 extracellular domains. Exons designated by tan boxes 1–4 encode extracellular ‘stalk domains’, immediately upstream from the transmembrane domain (TM). Stalk domains 1, 2 and 4 are substrates for distinct metalloproteases, which regulate NRG1 extracellular domain shedding. NRG1’s with stalk domain 3 contain a C-terminal stop codon before the TM and are secreted. Exons in the gray boxes encoding different cytoplasmic domains that also regulate extracellular domain shedding. Arrows: PCR primers used to analyze testicular Nrg1 variants. ( b ) Full-length Nrg1 transcripts amplified from undifferentiated type A spermatogonia encode variants of Type I, NRG1. Arrows: respective PCR primers used to clone Nrg1 variants. ( c ) Spermatogonia selectively express mRNAs encoding NRG1 and CSPG5 (qtPCR), n =cells from three different rats; ±S.E.M. Spermatogonia, Spermatocytes (differentiating spermatogonia/early spermatocytes), Tubular somatic cells and Interstitial somatic cells refer to Sg, Scy, IC and SC described in . ( d ) Western blots for NRG1 α 1 and NRG1 β 1 in the rat brain (B) and testis (T) and a primary spermatogonial line derived from undifferentiated type A spermatogonia (Sg). Arrows on left of each blot represent respective size molecular markers (kDa). Blue asterisks denote respective size NRG1 variants previously reported in the rat brain. ( e ) Immunolabeling for NRG1 α 1 (red cytoplasm) in adult rat testis sections. Nuclei counterstained with Hoechst 33342 dye (cyan). Pl=preleptotene spermatocyte; L=leptotene spermatocyte; eZ=early zygotene spermatocyte; P=mid-to-late pachytene spermatocytes; S8, S9, S12, S19=Step 8, 9, 12, 19 spermatids; VE=vascular endothelial cell; L=Leydig cell; S=Sertoli cell. Roman numerals denote spermatogenic stages. Scale bar, 100 μ m.

Article Snippet: Lapatinib (L-4804) was from LC Laboratories (Woburn, MA, USA); ERBB2-neutralizing IgG (AF1129), ERBB3 (348-RB) and ERBB4 (1131-ER) Fc chimeric proteins were from R&D Systems.

Techniques: Generated, Amplification, Western Blot, Derivative Assay, Immunolabeling

Journal: Cell Death Discovery

Article Title: NRG1 and KITL signal downstream of retinoic acid in the germline to support soma-free syncytial growth of differentiating spermatogonia

doi: 10.1038/cddiscovery.2015.18

Figure Lengend Snippet:

Article Snippet: Lapatinib (L-4804) was from LC Laboratories (Woburn, MA, USA); ERBB2-neutralizing IgG (AF1129), ERBB3 (348-RB) and ERBB4 (1131-ER) Fc chimeric proteins were from R&D Systems.

Techniques: