Ndrg1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

NDRG1 is a protein that plays a role in cellular stress response and cell growth regulation. It is commonly used as a marker to study these processes in biological research.

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Lab products found in correlation

Close spelling variants of this product

Anti-NDRG1 (10 citations) P-Ndrg1(T346), (8 citations) P-NDRG1 (6 citations) P-NDRG1-Thr346 (6 citations) Phospho-NDRG1 (5 citations) Phospho-NDRG1-Thr 346 (3 citations) Rabbit anti-NDRG1 (2 citations)

28 protocols using ndrg1

Western Blot Analysis of Signaling Proteins

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Western blot was performed using established procedures^{40 (link)}. Cells were lysed in lysis buffer (Beijing Dingguo Biotechnology Co. Ltd), incubated on ice for 30 min and centrifuged for 20 min to remove cell debris. Total cell lysates were subjected to SDS–polyacrylamide and immunoblotted with antibodies against EGFR (Sc-03-G; Santa Cruz), p44/42 ERK (9102; Cell Signaling), phospho-p44/42 ERK-Thr202/Tyr204 (9106; Cell Signaling), NDRG1 (5196; Cell Signaling), HER2 (Sc-7301; Santa-Cruz), β-actin (ab1801; Abcam).

Yang Y., Liu Y., Guo R., Fu Y., Zhang Z., Zhang P., Zhou P., Wang T., Huang T., Li X, & Li C. (2018). The novel dithiocarbamate, DpdtC suppresses HER2-overexpressed cancer cells by up-regulating NDRG1 via inactivation of HER2-ERK 1/2 signaling. Scientific Reports, 8, 3398.

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Antibody Profiling of mTOR Pathway

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The following antibodies were used in this study: 4EBP1 (catalog #9644, 1:4000 dilution), Phospho-4EBP1-Thr37/46 (#2855,1:1000), Phospho-4EBP1 -Ser65 (#9451,1:1000), p70S6 Kinase (#2708,1:1000), Phospho-p70S6 Kinase-Thr389 (#9234,1:1000), Phospho-S6-Ser240/244(#5364,1:2000), AKT(#4691,1:2000), Phospho-AKT-Ser473 (#4060,1:1000), EIF4E (#9742,1: 2000), EIF4G (#2498,1:1000), NDRG1(#5196,1:2000), Phospho-NDRG1-Thr 346(#5482,1:2000), Phospho-Tuberin/TSC2-Ser939 (#3615,1:1000), SGK3 (#85731:1000), Phospho-SGK3-Thr320 (#5642,1:500), Survivin (#2808, 1:1000) and Rictor (#2114,1:1000), and were purchased from Cell Signaling Technology (Danvers, MA). In addition, γ-Tubulin (sc-7396,1:500) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tuberin (ab32554,1:1000) and INPP4B (ab81269, 1:1000) were purchased from Abcam (Cambridge, UK).

Wang H., Huang F., Zhang Z., Wang P., Luo Y., Li H., Li N., Wang J., Zhou J., Wang Y, & Li S. (2019). Feedback Activation of SGK3 and AKT Contributes to Rapamycin Resistance by Reactivating mTORC1/4EBP1 Axis via TSC2 in Breast Cancer. International Journal of Biological Sciences, 15(5), 929-941.

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Immunoblotting Analysis of Cell Signaling

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Cells were harvested and protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad, Munich, Germany). We subjected 20 µg acquired protein to SDS page analysis. After washing with tris-buffered saline with Tween20 and blocking in 5% skim milk, the resulting membranes were incubated overnight with antibodies to NDRG1, P-NDRG1 (Thr346), S6RP, P-S6RP (Ser240/44), 4EBP1, P-4EBP1, p62, p-ULK (all from Cell Signaling, Cambridge, UK) and LC-3 (Sigma). Following one hour of incubation with secondary rabbit or goat antibody (Santa Cruz Biotechnology, Dallas, TX, USA), chemiluminescence was used for detection.

Heinzen D., Divé I., Lorenz N.I., Luger A.L., Steinbach J.P, & Ronellenfitsch M.W. (2019). Second Generation mTOR Inhibitors as a Double-Edged Sword in Malignant Glioma Treatment. International Journal of Molecular Sciences, 20(18), 4474.

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Epigenetic Regulation of Neuroendocrine Markers in PCa Cells

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PCa cells were cultured in RPMI 1640 with 10% FBS and 1% P/S. The cells were treated with vehicle or 5-Aza (5µM) for 10 days at 37°C. Whole cell lysates were prepared from cells, separated on 4–20% Tris-Glycine gel and transferred to PVDF membranes. The membranes were incubated with 5% milk for 1 hour and incubated with primary antibodies overnight at 4°C. Primary antibodies used were as follows: N-Myc downstream regulated 1 (NDRG1, 1:1,000 dilution, cat. 9485, Cell Signaling,), Synaptophysin (1:1,000 dilution, cat. 4329, Cell Signaling,), Enolase-2 (ENO2, 1:1,000 dilution, cat. 9536, Cell Signaling,), H3K9me3 (1:1,000 dilution, cat. 13969, Cell Signaling), and H3K27me3 (1:1,000 dilution, cat. 9733, Cell Signaling). Blots were incubated with peroxidase-coupled anti-mouse IgG secondary antibody (cat. 7076, 1:2,000 dilution, Cell Signaling) or peroxidase-coupled anti-rabbit IgG secondary antibody (cat. 7074, 1:2,000 dilution, Cell Signaling) for 1 hour, and protein expression was detected with SuperSignal West Dura Chemiluminescent Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). Membranes were reprobed with monoclonal anti-β-Actin antibody (1:1,000 dilution, cat. 4970, Cell Signaling) to control for equal loading.

Lee E., Wang J., Jung Y., Cackowski F.C, & Taichman R.S. (2018). Reduction of Histone Marks, H3K9me3 and H3K27me3 by Epidrug Induces Neuroendocrine Differentiation in Prostate Cancer. Journal of cellular biochemistry, 119(4), 3697-3705.

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Erlotinib Signaling Pathway Analysis

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Erlotinib (#S1023) was obtained from Selleck Chemicals (Houston, TX, USA). Puromycin (#A1113803) was purchased from Life Technologies (Carlsbad, CA, USA). Hoechst 33342 (#H21492) and propidium iodine (#P1304MP) were acquired from Invitrogen (Waltham, MA, USA). The Rictor (#2114), phospho-tyrosine (#5465), phospho-NDRG1 (T346; #5482), NDRG1 (#9485), phospho-Akt (S473; #4060), Akt (#9272), phospho-S6 (S240/4; #2215), S6 (#2217), phospho-4EBPl (T37/46; #2855), 4EBP1 (#9452), phospho-EGFR (Y1068; #3777), EGFR (#4267) and MET (#8198) antibodies were from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against β-actin (#A1978) were purchased from Sigma (St. Louis, MO, USA).

Chiang C.T., Demetriou A.N., Ung N., Choudhury N., Ghaffarian K., Ruderman D.L, & Mumenthaler S.M. (2018). mTORC2 contributes to the metabolic reprogramming in EGFR tyrosine-kinase inhibitor resistant cells in non-small cell lung cancer. Cancer letters, 434, 152-159.

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Protein Analysis Using SDS-PAGE and Immunoblotting

Cited 1 time

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SDS-PAGE and immunoblotting were carried out as previously described (10 (link)). Cells were lysed with either radioimmunoprecipitation assay lysis buffer as previously described (10 (link)) or 1% Triton X-100 lysis buffer as described (46 (link)). Antibodies recognizing ERRB3 Y1197, ERBB3, EPHA2 S897, NDRG1 T346, Akt S473, Akt T308, Akt, p70S6K T389, p70S6K, ribosomal S6 S240/244, S6, IGF1R Y1135/36/IR Y1150/51, IGF1R, FOXO1/3a T24/T32, and FOXO3 were from Cell Signaling. Other antibodies included EPHA2 (Santa Cruz), secreted form of NRG1 (R&D Systems), NDRG1 (Abcam), and GAPDH (EMD Millipore). Anti-NF2/merlin polyclonal antibody has been previously described (61 (link)).

Beauchamp R.L., Erdin S., Witt L., Jordan J.T., Plotkin S.R., Gusella J.F, & Ramesh V. (2020). mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition. The Journal of Biological Chemistry, 296, 100157.

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Western Blot Analysis of CIT, NDRG1, and GAPDH

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Western blot was done using BioRad pre-casted gels and a trans-turbo transfer machine (BioRad). The following are the antibodies used in this study: CIT antibody (BD Biosciences # 611376), NDRG1 (Cell Signaling # 9485), phospho-NDRG1 (Cell Signaling # 5482) and GAPDH (Sana Cruz # SC32233).

Davies R., Liu L., Taotao S., Tuano N., Chaturvedi R., Huang K.K., Itman C., Mandoli A., Qamra A., Hu C., Powell D., Daly R.J., Tan P, & Rosenbluh J. (2021). CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies. Genome Biology, 22, 47.

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Compound C and Sodium Palmitate Signaling Pathway Analysis

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Compound C was obtained from Enzo Life Sciences. Sodium Palmitate is from NU-CHEK Prep Inc. For western blotting, anti-Flag M2 (RRID:AB_259529) antibody was purchased from Sigma, antibodies against SGK1 (RRID:AB_2687476), SGK2 (RRID:AB_10828732), SGK3 (RRID:AB_10949507), HSP90 (RRID:AB_2233307), Akt (RRID:AB_915783), p-Akt (Thr308) (RRID:AB_2255933), p-Akt (Ser473) (RRID:AB_2315049), NDRG1 (RRID: AB_11140640), p-NDRG1(Thr346) (RRID:AB_10693451), FoxO1 (RRID:AB_2106495), p-FoxO1/3 (Thr24/32) (RRID:AB_2106814), S6K (RRID:AB_390722), p-S6K (Thr389) (RRID:AB_2269803), S6 (RRID:AB_331355), p-S6 (S240/244) (RRID:AB_10694233), 4EBP1 (RRID:AB_2097841), p-4EBP1 (Thr37/46) (RRID:AB_560835), p-4EBP1 (Ser65) (RRID:AB_330947), AMPKα (RRID:AB_10624867), p-AMPKα (Thr172) (RRID:AB_331250), p-AMPKα(Ser485/491) (RRID:AB_331250), ACC1 (RRID:AB_2219397), p-ACC1 (Ser79) (RRID:AB_330337), Raptor (RRID:AB_561245)and p-Raptor (Ser792) (RRID:AB_2249475) were obtained from Cell Signaling Technology. Anti-actin (C4) was obtained from Abcam.

Zhou B., Zhang Y., Li S., Wu L., Fejes-Toth G., Naray-Fejes-Toth A, & Soukas A.A. (2021). Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase. Cell reports, 37(1), 109785.

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Comprehensive Antibody Panel for SGK1, AKT, Chk1, and NDRG1 Signaling

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Anti-SGK1 (#12103), AKT (#4691), Phospho-AKT-Ser473 (#9271), Phospho-AKT-Thr308 (#13038), Chk1(#2360), Phospho-Chk1-Ser345 (#2348), Phospho-Histone H2A.X-Ser139 (#2577), NDRG1(#9395), Phospho-NDRG1-Thr346 (#3217), Phospho-NEDD4L-Ser448 (#8063), Hsp90 (#4877), CDC37(#4793), HA Tag (#3724), and FLAG Tag (#14793) were from Cell Signaling Technology. Phospho-SGK1-S422 (#44-1264G), FAM122A (#MA5-24510), V5-HRP (#R961-25) were from Invitrogen; NEDD4-2 (#ab131167) from Abcam; PP5 antibody (#sc-271816) from Santa Cruz Biotechnology; FAM122A (#NBP2-31646) from Novus Biologicals; GAPDH-HRP (#BE0034-100) from Easybio; actin-HRP (#AM8502b) from ABGENT; and secondary antibodies, anti-rabbit IgG HRP (#A16110), and anti-mouse IgG HRP (#A16072) from Invitrogen.

Gu W., Zheng H, & Canessa C.M. (2023). Phosphatases maintain low catalytic activity of SGK1: DNA damage resets the balance in favor of phosphorylation. The Journal of Biological Chemistry, 299(8), 104941.

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Antibody Profiling of Cell Signaling

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Antibodies against the following proteins were used in this study: TMBIM6/BI-1 (1:100, ab51905), and RPL19 (1:1000, ab128648) (all from Abcam, Cambridge, UK); RICTOR (1:2000, A300-459A) (Bethyl Laboratories, Montgomery, TX, USA); AKT (1:1000, #9272), GST (1:1000, #2622), mTOR (1:1000, #2972), mTOR (1:1000, #4517), NDRG1 (1:1000, #9408), phospho-Ser473-AKT (1:1000, #9271), phospho-Ser939-TSC2 (1:1000, #3615), phospho-Thr308-AKT (1:1000, #4056), TSC2 (1:1000, #4308), phospho-Thr346-NDRG1 (1:1000, #3217), SIN1 (1:1000, #12860), p70 S6 Kinase (1:100, #9202), RICTOR (1:1000, #2114), RICTOR (Sepharose bead conjugate, #5379), and RAPTOR (1:1000, #2280) (all from Cell Signaling Technology, Danvers, MA, USA); HA (1:2000, 11867423001) (Roche Diagnostics, Basel, Switzerland); actin (1:1000, sc-47778), Ki-67 (1:100, sc-15402), phospho-Thr450-AKT (1:1000, sc-293094), RPL19 (1:1000, sc-100830), and RPS16 (1:1000, sc-102087), (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies (1:10,000) for immunoblotting (Jackson ImmunoResearch, West Grove, PA, USA) and immunoprecipitation (1:5000, sc-2006) (Santa Cruz Biotechnology) were also used. Insulin (I3769), EGF (E9644), and IGF1 (I3769) were from Sigma-Aldrich. BAPTA-AM (B6769), BAPTA (B1212), and EGTA-AM (E1219) were from Thermo Fisher Scientific (Waltham, MA, USA).

Kim H.K., Bhattarai K.R., Junjappa R.P., Ahn J.H., Pagire S.H., Yoo H.J., Han J., Lee D., Kim K.W., Kim H.R, & Chae H.J. (2020). TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation. Nature Communications, 11, 4012.

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