nrg 1 antibody  (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: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

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: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

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: Spermatogonial culture and differentiation media

Article Snippet: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

Techniques:

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: NRG1/ERBB3/ERBB2-dependent growth of differentiating spermatogonia. ( a ) ATRA and Neuregulin-1 stimulate development of aligned spermatogonia. tg GCS-EGFP + spermatogonial scored/well after 1 week of culture in SG F Medium on laminin and then 6 additional days with and without ATRA (3 μ M) in combination with GDNF (0.1 nM) and/or NRG1 (5 nM). Representative of duplicate in vitro spermatogenesis colony-forming assays scored after 2 days in SG F Medium and 6 days in SD Medium (±S.E.M., triplicate wells). ( b ) Effects of various ERBB-family receptor agonists on soma-free rat spermatogonial cultures on laminin. Respective ERBB agonists were substituted for NRG1 in SD Medium. Representative of duplicate in vitro spermatogenesis colony forming assays (±S.E.M., triplicate wells). Inset: western blotting for pAKT-S473 (arrowhead) and p42/44 (lower bands) in spermatogonia in SG F Medium for 2 days, prior to culturing with and without ATRA for 4 days, and then treating with and without NRG1 for 20 min before lysing. ( c ) ERBB2 inhibitors disrupt NRG1-dependent spermatogonial development on laminin. Representative of duplicate colony-forming assays scored (±S.E.M., triplicate wells). ( d ) Images of NRG1-dependent syncytial growth of tgGCS-EGFP + spermatogenic cells on laminin. Scale, 100 μ m. Nuclei labeled with Hoechst 33342 dye.

Article Snippet: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

Techniques: In Vitro, Western Blot, Labeling

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: KITL supports spermatogonial differentiation independent of ERBB2. ( a ) Images of NRG1- and KITL-dependent spermatogonial differentiation after 2 days in SG F Medium and 6 days in SD Medium. Spermatogonia are labeled with the germ cell marker DAZL (green); nuclei labeled with Hoechst 33342 dye. Scale, 100 μ m. ( b ) Spermatogonia cultured as in panel ( a ) labeled with antibodies to the germ cell marker TEX14 (green) and a marker for undifferentiated type A spermatogonia, ZBTB16 (red); nuclei labeled with Hoechst 33342 dye. Scale, 50 μ m. ( c ) ERBB2 inhibitors selectively disrupt NRG1-dependent but not KITL-dependent spermatogonial development on laminin. Representative of duplicate colony-forming assays (±S.E.M., triplicate wells). ( d ) KITL rescues spermatogonial syncytial growth of Erbb3 -deficient germlines on laminin. Representative of duplicate colony-forming assays (±S.E.M., triplicate wells).

Article Snippet: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

Techniques: Labeling, Marker, Cell Culture

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: Spermatogonia cultured in SD Medium lose germline stem cell activity. ( a ) Diagram of spermatogonial culture experiment on laminin matrix prior to transplanting respective spermatogonial cultures into recipient rat testes ( n =5 rats/condition with right testis transplanted; P <0.0001 control versus each test condition, multiple t -tests). ( b ) Left: Relative numbers of spermatogenic colonies generated/testis/1000 donor germ cells (tg GCS-EGFP + ) harvested from cultures described in panel ( a ) after 1 week in respective SD Media containing NRG1 and/or KITL (D4–D11). Donor ‘Control’ cultures were maintained on laminin in SG Medium the full 10 days (D1–D11) prior to transplantation. Recipients analyzed 28 days posttransplantation. Right: Images of recipient rat testes after being transplanted with spermatogonia from each respective culture condition. Scale, 1 cm.

Article Snippet: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

Techniques: Cell Culture, Activity Assay, Control, Generated, Transplantation Assay

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: Soma-independent development of differentiating spermatogonia. FGF2 and GDNF support spermatogonial stem cell renewal and syncytial growth of early undifferentiated spermatogonial progenitors (DAZL + , TEX14 + , ZBTB16 + ) in culture on laminin. Retinoic acid acts in the germline to drive transformation of undifferentiated spermatogonia into nascent differentiating spermatogonia. Polypeptide growth factors NRG1 and KITL are required for survival of differentiating spermatogonia on laminin without somatic cells. In the absence of NRG1 and KITL, ATRA drives undifferentiated spermatogonia to undergo development into syncytia of ~4-16 DAZL + , TEX14 + , ZBTB16 − spermatogenic cells that degenerate.

Article Snippet: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

Techniques: Transformation Assay

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: Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1 α 1 (R&D Systems, Inc., cat. no. AF296NA), NRG1 β 1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC).

Techniques: