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Neurod1

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NEUROD1 is a transcription factor that plays a role in the development and differentiation of neurons. It is involved in the regulation of genes related to neuronal function and is important for the survival and maturation of neurons.

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Lab products found in correlation

P akt, by Cell Signaling Technology (2 mentions) Ascl1, by BD (2 mentions) Erk1 2, by Cell Signaling Technology (2 mentions) Snail, by Abcam (2 mentions) Glial fibrillary acidic protein (gfap), by Merck Group (2 mentions) β catenin, by Cell Signaling Technology (1 mentions) Vinculin, by Cell Signaling Technology (1 mentions) Pou3f2, by Cell Signaling Technology (1 mentions) P pras40, by Cell Signaling Technology (1 mentions) Sc 1657, by Santa Cruz Biotechnology (1 mentions) Hoechst, by Merck Group (1 mentions) Herc2, by BD (1 mentions) Sc 2025, by Santa Cruz Biotechnology (1 mentions) Goat serum, by Merck Group (1 mentions) Ha mms 101p, by Fortrea (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions) Iba 1, by Abcam (1 mentions) Cleaved caspase 3, by Abcam (1 mentions)

Spelling variants of this product

Anti-NeuroD1 (6 citations) NEUROD1 (clone EPR17084, (3 citations) Rabbit anti-NeuroD1 antibody (2 citations) Mouse anti-NEUROD1 (2 citations)

14 protocols using neurod1

1

Protein Expression Analysis by Western Blot

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Protein extraction and western blot were performed as previously described(68 (link)). Antibodies for FOXN4 (#PA539174, ThermoFisher), ONECUT2 (#ab28466, Abcam), POU3F2 (#12137, Cell Signaling Technology), EGFR (#4267, Cell Signaling Technology), ASCL1 (#556604, BD), NEUROD1 (#ab109224, Abcam), pAKT (S473, #4060S, Cell Signaling Technology), pPRAS40 (T246, #13175, Cell Signaling Technology), Beta catenin (#8480, Cell Signaling Technology), Vinculin (#13901, Cell Signaling Technology) and actin (#3700, Cell Signaling Technology) were used.
Quintanal-Villalonga A., Taniguchi H., Zhan Y.A., Hasan M.M., Chavan S.S., Meng F., Uddin F., Manoj P., Donoghue M.T., Won H.H., Chan J.M., Ciampricotti M., Chow A., Offin M., Chang J.C., Ray-Kirton J., Tischfield S.E., Egger J., Bhanot U.K., Linkov I., Asher M., Sinha S., Silber J., Iacobuzio-Donahue C.A., Roehrl M.H., Hollmann T.J., Yu H.A., Qiu J., de Stanchina E., Baine M.K., Rekhtman N., Poirier J.T., Loomis B., Koche R.P., Rudin C.M, & Sen T. (2021). Multi-omic analysis of lung tumors defines pathways activated in neuroendocrine transformation. Cancer discovery, 11(12), 3028-3047.
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2

Antibody Validation Protocols for Cell Biology

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Antibodies to GluR2 (75–002, NeuroMab), Bassoon (ADI-VAM-PS003-F, Enzo Life Sciences), DsRed (632496, Clonetech), 14-3-3 (sc-1657, Santa Cruz Biotechnology), Tubulin (T5326, Sigma), NeuroD1 (ab60704, Abcam), HERC2 (612366, BD Bioscience), Flag (A2220, Sigma M2), ERK1/2 (9102, Cell Signaling), Hoechst (B2883, Sigma), Cre (69050, Millipore), UBC13 (37-1100, Invitrogen), HA (MMS-101P, Covance), Parp (Cell Signaling), GFP (A6455, Invitrogen/ab3970, Abcam), goat serum (G9023, Sigma), rabbit IgG (12-370, Millipore), and mouse IgG (sc-2025, Santa Cruz) were purchased. The NEURL4 antibody was kindly provided by Brian Dynlacht (NYU Cancer Institute).
A rabbit RNF8 antibody was raised against bacterially produced glutathione-S-transferase fusion protein containing 100–250 aa of RNF8 and purified by affinity chromatography.
Uncropped western blot images are shown in Supplementary Fig. 11.
Valnegri P., Huang J., Yamada T., Yang Y., Mejia L.A., Cho H.Y., Oldenborg A, & Bonni A. (2017). RNF8/UBC13 ubiquitin signaling suppresses synapse formation in the mammalian brain. Nature Communications, 8, 1271.
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3

Molecular Mechanisms of Inflammatory Response

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Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were provided by Corning (New York, NY, USA). Heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) were purchased from Gibco (Life Technologies, Gaithersburg, MD, USA). LPS was obtained from Sigma-Aldrich (St. Louis, MO, USA). TNF-α and IL-6 were measured using TNF-α and IL-6 DuoSet ELISA kits (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Reactive oxygen species (ROS) production was measured using H2DCF-DA from Molecular Probes (Eugene, OR, USA). Antibodies against ERK1/2, p-ERK1/2, p38, p-p38, NF-κB, NLRP3, COX-2, iNOS, rabbit IgG HRP, and mouse IgG HRP were purchased from Cell Signaling Technology (Danvers, MA, USA); NeuroD1, Iba-1, GFAP, cleaved caspase-3, mouse IgG H&L (Alexa Fluor 488), mouse IgG H&L (Alexa Fluor 568), mouse IgG H&L (Alexa Fluor 488), rabbit IgG H&L (Alexa Fluor 488) were purchased from Abcam (Cambridge, UK); and JNK, p-JNK, caspase-1 (p20), ASC, p-STAT-3, β-actin, and Lamin-B1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Choi H.R., Ha J.S., Kim E.A., Cho S.W, & Yang S.J. (2022). MiR-30a-5p and miR-153-3p regulate LPS-induced neuroinflammatory response and neuronal apoptosis by targeting NeuroD1. BMB Reports, 55(9), 447-452.
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4

Comprehensive Protein Expression Analysis in Stem Cell Research

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Antibodies against octamer-binding transcription factor 4 (1:1000, Oct-4; ab200834, abcam), Brachyury (1:1000, sc-166962, Santa Cruz Biotechnology), GATA-binding protein 4 (1:1000, GATA4; sc-25310, Santa Cruz Biotechnology), SRY box transcription factor 17 (1:1000, Sox17; AF1924, R&D system), neuronal differentiation 1 (1:1000, NeuroD1; ab205300, abcam), XRCC5 (1:2000, AF5819, R&D system), IGF2BP1 (1:1000, ab184305, abcam), RBBP4 (1:1000, MAB7416, R&D system), NPM1 (1:1000, sc-271737, Santa Cruz Biotechnology), PCNA (1:1000, sc-56, Santa Cruz Biotechnology), GNAI2 (1:1000, ab137050, abcam), PHB2 (1:1000, sc-133084, Santa Cruz Biotechnology), LAMB1 (1:1000, sc-17810, Santa Cruz Biotechnology), LAMC1 (1:1000, sc-17751, Santa Cruz Biotechnology), β-catenin (1:1000, sc-7963, Santa Cruz Biotechnology), AKT (1:2000, #4691, Cell Signaling Technology), Jun amino-terminal kinases (JNK) (1:1000, #9252, Cell Signaling Technology), phosphorylated (p)-JNK (1:1000, #9255, Cell Signaling Technology), p38 (1:1000, sc-81621, Santa Cruz Biotechnology), p-p38 (1:1000, #9216, Cell Signaling Technology), TGF-β (1:1000, #3711, Cell Signaling Technology), CTGF (1:1000, sc-385970, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; (1:3000, sc-47724, Santa Cruz Biotechnology) were employed for the western blot analysis.
Seo J.H, & Jeon Y.J. (2021). Global Proteomic Analysis of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells via Connective Tissue Growth Factor Treatment under Chemically Defined Feeder-Free Culture Conditions. Journal of Microbiology and Biotechnology, 32(1), 126-140.
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5

Protein Expression Profiling of Cell Populations

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Adherent and suspension cell populations were collected and washed in PBS prior to lysis in RIPA buffer (Sigma) with protease inhibitor cocktails (Roche, South San Francisco, CA, USA) plus PMSF. Cell lysates were briefly sonicated prior and precleared by centrifugation. 30 µg of protein was loaded on 4–12% SDS/PAGE and transferred to nitrocellulose membranes. Samples were incubated overnight at 4 °C in primary antibodies and 1 h at room temperature in secondary antibodies. Imaging was performed using LiCor Imaging System (Lincoln, NE, USA).
Antibodies used in this study are KDM1A (Millipore cs207350, Bethyl, Montgomery, TX, USA, A300‐215A), NSE (CST, Danvers, MA, USA, 9536), SYP (CST 12270), GRP (Sigma HPA007314), NCAM (CST 3576), CHGA (Abcam, Branford, CT, USA, 45179), FOXA2 (CST 3143), OVOL2 (Abcam 129161), LEF1 (CST 2286), ASCL1 (Abnova, Walnut, CA, USA, H0429‐M02), SOX2 (CST 4900), INSM1 (Abcam 170876), NEUROD1 (117562), SMAD3 (CST 9513), NFKB2 (CST 3017), CMYC (CST 9402), MITF (Abcam 140606), SNAIL (CST 3895), VIM (CST 5741), CDH2 (CST 14215), CDH1 (CST 3195P), and ZEB1 (CST 3396P).
Yan W., Chung C., Xie T., Ozeck M., Nichols T.C., Frey J., Udyavar A.R., Sharma S, & Paul T.A. (2021). Intrinsic and acquired drug resistance to LSD1 inhibitors in small cell lung cancer occurs through a TEAD4‐driven transcriptional state. Molecular Oncology, 16(6), 1309-1328.
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6

Neuronal Differentiation Protocols

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The materials used in this study's experiments are listed below along with their respective suppliers: Lithium chloride, Sodium butyrate, Trichostatin A, and Valproic acid from Sigma (St. Louis, MO); ECL™ reagents from Amersham Life Science (Arlington Heights, IL); Trizol® reagent, SuperScript™II Reverse Transcriptase, Tert siRNA [TERT-RSS313560 (rat), TERT-MSS211505-7 (mouse)] from Invitrogen (Carlsbad, CA); Protein G Agarose from Millipore (Billerica, MA); TDZD-8 (4-Benzyl-2-methyl-1,2,4,-Thiadiazolidine-3,5-dione) from Calbiochem (La Jolla, CA); Taq polymerase from Takara (Shiga, Japan).
Antibodies from the following companies: β-actin antibody from Sigma (St. Louis, MO); Tuj-1 antibody from Covance (Princeton, NJ); TERT antibody from Santa Cruz Biotechnology (Santa Cruz, CA); vGluT1, PSD-95, α-CaMKII, NeuroD1, Mash1, BRG1, Synaptophysin antibody from Abcam (Cambridgeshire, England); GAD, Nestin, GFAP, Pax6 and Ngn2GFAP antibody from Millipore (Billerica, MA); HDAC1, Histone H3, Acetyl-Histone H3, GSK3β and phospho-GSK3β antibody from Cell signaling (Boston, MA).
Kim K.C., Choi C.S., Gonzales E.L., Mabunga D.F., Lee S.H., Jeon S.J., Hwangbo R., Hong M., Ryu J.H., Han S.H., Bahn G.H, & Shin C.Y. (2017). Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development. Experimental Neurobiology, 26(5), 252-265.
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7

Immunohistochemical Analysis of Mouse Brain

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Mice were euthanized and perfused with intracardial injection of 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS). Brains were isolated and post-fixed overnight with 4% (w/v) paraformaldehyde at 4 °C. After cryoprotection with 30% sucrose in PBS for 72 h at 4 °C, 40-μm brain slices were collected through a sliding microtome (Leica). For immunostaining, brain sections were treated with 50% formamide in 1 X SSC buffer for 2 h at 65 °C. For BrdU staining, the sections were pretreated with 2N HCl for 30 min at 37 °C. The following primary antibodies were used: GFP (Chicken; 1:2,000; Aves Labs), mCherry (Goat; 1:2,000; Mybiosource), NeuN (Rabbit; 1:10,000; Abcam), GFAP (Mouse; 1:2,000; Sigma), Cre (Rabbit; 1:500; Covance), ALDH1L1 (Mouse; 1:200; Neuromab), ALDOC (Goat; 1:100; Santa Cruz), BrdU (Rat; 1:500; Bio-Rad), NEUROD1 (Rabbit; 1:1,000; Abcam), PTBP1 (Rabbit, 1:1,000, ABclonal), FLAG (Rabbit; 1:300; Sigma), HA (Chicken; 1:300; Aves), DCX (Goat; 1:500; Santa Cruz), and MAP2 (Mouse; 1:1,000; Proteintech). Alexa Fluor 488-, 555-, or 647-conjugated corresponding secondary antibodies from Jackson ImmunoResearch were used for indirect fluorescence (1:2,000). Nuclei were counterstained with Hoechst 33342 (Hst). Images were captured using a Zeiss LSM700 or Nikon A1R confocal microscope for analysis.
Wang L.L., Serrano C., Zhong X., Ma S., Zou Y, & Zhang C.L. (2021). Revisiting astrocyte to neuron conversion with lineage tracing in vivo. Cell, 184(21), 5465-5481.e16.
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8

Quantification and Immunoblotting of SCLC Markers

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SCLC cells were counted before lysis with Laemmli sample buffer (Bio-Rad). One-million cells were lysed with 200 µL sample buffer containing 2-mercaptoethanol and boiled for 10 min. Equal volumes of samples were separated by SDS-PAGE gel followed by transfer to nitrocellulose membrane and immunoblotting. Primary antibodies used in this study included POU2F3 (1:1000; Sigma, HPA019652), ASCL1 (1:1000; BD Biosciences, clone 24B72D11.1 ), and NEUROD1 (1:1000; Abcam, ab60704).
Huang Y.H., Klingbeil O., He X.Y., Wu X.S., Arun G., Lu B., Somerville T.D., Milazzo J.P., Wilkinson J.E., Demerdash O.E., Spector D.L., Egeblad M., Shi J, & Vakoc C.R. (2018). POU2F3 is a master regulator of a tuft cell-like variant of small cell lung cancer. Genes & Development, 32(13-14), 915-928.
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9

Protein Expression Analysis of SCLC

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Fresh-frozen xenograft samples were lysed in RIPA buffer using a TissueLyzer II (Qiagen) homogenizer. SDS-PAGE was performed by standard methods, and PVDF membranes were probed with the following antibodies: from Cell Signaling Technology: alpha-tubulin, beta-actin, NeuroD1, total EGFR, pAKT (S473), pERK1/2 (T202/Y204); from Abcam: Ascl1 (MASH1); from Santa Cruz Biotechnology: Slfn11. Membranes were imaged with a Syngene G:BOX, and band densitometry was performed using Syngene GeneSys software. Ratio to loading control (alpha-tubulin) was calculated, and lysates from established SCLC cell lines (CORL88, CORL279, NCIH82, NCIH1048, and DMS273) were used as interblot standards. Cell lines were obtained between 2015 and 2017 from the MGH Center for Molecular Therapeutics, which performs routine authentication by single-nucleotide polymorphism and short tandem repeat analyses, and were passaged in HITES media + 2% FBS for less than 3 months prior to lysate preparation.
Drapkin B.J., George J., Christensen C.L., Mino-Kenudson M., Dries R., Sundaresan T., Phat S., Myers D.T., Zhong J., Igo P., Hazar-Rethinam M.H., Licausi J.A., Gomez-Caraballo M., Kem M., Jani K.N., Azimi R., Abedpour N., Menon R., Lakis S., Heist R.S., Büttner R., Haas S., Sequist L.V., Shaw A.T., Wong K.K., Hata A.N., Toner M., Maheswaran S., Haber D.A., Peifer M., Dyson N., Thomas R.K, & Farago A.F. (2018). Genomic and Functional Fidelity of Small Cell Lung Cancer Patient-Derived Xenografts. Cancer discovery, 8(5), 600-615.
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10

Immunohistochemical Subtyping for SCLC

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All cases were stained for ASCL1 (clone: 24B72D11.1; BD Biosciences), NEUROD1 (clone: EPR17084; Abcam), and POU2F3 (clone: polyclonal; Atlas Antibodies) for subtyping. The nuclear expression of each marker was semiquantitatively recorded for percentage of positive cells (0%–100%) and intensity of staining (0 = no stain, 1 = weak, 2 = moderate, 3 = strong). A combination H score (range 0–300) was calculated as previously described: H score = (0 × % negative) + (1 × % weakly positive) + (2 × % moderately positive) + (3 × % strongly positive cells).20 (link) Marker with H score less than 50 was interpreted as negative, as previously suggested.9 (link) Each slide was scored by two pathologists independently. Average scores were calculated and recorded for analysis. Cases with substantial discrepancy leading to subtype difference were re-reviewed by both pathologists together until consensus was reached.
Lo Y.C., Rivera-Concepcion J., Vasmatzis G., Aubry M.C, & Leventakos K. (2023). Subtype of SCLC Is an Intrinsic and Persistent Feature Through Systemic Treatment. JTO Clinical and Research Reports, 4(9), 100561.
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