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plasmid pcdna3 flag rheb n153t  (Addgene inc)


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    Structured Review

    Addgene inc plasmid pcdna3 flag rheb n153t
    Plasmid Pcdna3 Flag Rheb N153t, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pcdna3+flag+rheb+n153t/pcDNA3-FLAG-Rheb-N153T+(Plasmid+%2319997)/pmc09648415-88-20-22
    Average 91 stars, based on 14 article reviews
    plasmid pcdna3 flag rheb n153t - by Bioz Stars, 2026-09
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    Related Articles

    Transfection:

    Article Title: VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle
    Article Snippet: The cells were serum starved (no serum in the growth media) in Hanks’ Balanced Salt Solution (HBSS; Invitrogen, 14025076) for 1 h then restimulated with the DMEM-FBS full medium containing 10% FBS. .. For transfection, U2OS cells were cultured on coverslips and transfected with 1 μg each of pcDNA3-FLAG-RHEB-N153T (Addgene, plasmid 19997; depositing Lab: Fuyuhiko Tamanoi) and a GFP control plasmid pEGFPN1 (Clontech, 6085–1) using Lipofectamine 2000 (Invitrogen, 11668–019) according to the manufacturer’s instructions. .. Immunoblot analysis Muscle tissues and U2OS cells were homogenized in Radioimmunoprecipitation assay lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% NP-40 [Sigma, I3021], 0.25% Na deoxycholate [Sigma-Aldrich, 30970], 1 mm ethylenediaminetetraacetic acid) supplemented with protease inhibitor cocktail (Sigma-Aldrich, S8820).

    Article Title: Mitochondria are required for pro‐ageing features of the senescent phenotype
    Article Snippet: .. For the overexpression of activated Rheb and PGC‐1β, MEFs were transfected with an empty vector and pcDNA3‐flag‐Rheb‐N153T (Addgene #19997) or pcDNAf:PGC‐1β (Addgene #1031) respectively, using Lipofectamine TM 2000 (Invitrogen, 11668‐019) following the manufacturer's instructions. .. YFP‐Parkin retroviral production: phoenix amphotropic cells were incubated for 24 h in antibiotic‐free medium before proceeding to transfection with a LZRS or LZRS‐YFP_Parkin vector using Lipofectamine TM 2000 (Invitrogen, 11668‐019) following the manufacturer instructions.

    Cell Culture:

    Article Title: VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle
    Article Snippet: The cells were serum starved (no serum in the growth media) in Hanks’ Balanced Salt Solution (HBSS; Invitrogen, 14025076) for 1 h then restimulated with the DMEM-FBS full medium containing 10% FBS. .. For transfection, U2OS cells were cultured on coverslips and transfected with 1 μg each of pcDNA3-FLAG-RHEB-N153T (Addgene, plasmid 19997; depositing Lab: Fuyuhiko Tamanoi) and a GFP control plasmid pEGFPN1 (Clontech, 6085–1) using Lipofectamine 2000 (Invitrogen, 11668–019) according to the manufacturer’s instructions. .. Immunoblot analysis Muscle tissues and U2OS cells were homogenized in Radioimmunoprecipitation assay lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% NP-40 [Sigma, I3021], 0.25% Na deoxycholate [Sigma-Aldrich, 30970], 1 mm ethylenediaminetetraacetic acid) supplemented with protease inhibitor cocktail (Sigma-Aldrich, S8820).

    Plasmid Preparation:

    Article Title: VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle
    Article Snippet: The cells were serum starved (no serum in the growth media) in Hanks’ Balanced Salt Solution (HBSS; Invitrogen, 14025076) for 1 h then restimulated with the DMEM-FBS full medium containing 10% FBS. .. For transfection, U2OS cells were cultured on coverslips and transfected with 1 μg each of pcDNA3-FLAG-RHEB-N153T (Addgene, plasmid 19997; depositing Lab: Fuyuhiko Tamanoi) and a GFP control plasmid pEGFPN1 (Clontech, 6085–1) using Lipofectamine 2000 (Invitrogen, 11668–019) according to the manufacturer’s instructions. .. Immunoblot analysis Muscle tissues and U2OS cells were homogenized in Radioimmunoprecipitation assay lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% NP-40 [Sigma, I3021], 0.25% Na deoxycholate [Sigma-Aldrich, 30970], 1 mm ethylenediaminetetraacetic acid) supplemented with protease inhibitor cocktail (Sigma-Aldrich, S8820).

    Article Title: Regulation of mTORC1 by lysosomal calcium and calmodulin
    Article Snippet: Human GAPDH VIC (Hs02758991_g1) was used as an endogenous control. .. Myc-mTOR (Addgene plasmid # 1861), pRK5-HA GST RagA 66L (Addgene plasmid # 19300), pRK5-HA GST RagC 75L (Addgene plasmid # 19305) and HA GST PreScission p70 S6K1 (Addgene plasmid # 15511) were gifts from David Sabatini. pcDNA3-FLAG-Rheb-N153T (Addgene plasmid # 19997) was a gift from Fuyuhiko Tamanoi. pcDNA-CaM was a gift from David Yue. .. TRPML1-HA (Addgene plasmid # 18825) was a gift from Craig Montell.

    Control:

    Article Title: VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle
    Article Snippet: The cells were serum starved (no serum in the growth media) in Hanks’ Balanced Salt Solution (HBSS; Invitrogen, 14025076) for 1 h then restimulated with the DMEM-FBS full medium containing 10% FBS. .. For transfection, U2OS cells were cultured on coverslips and transfected with 1 μg each of pcDNA3-FLAG-RHEB-N153T (Addgene, plasmid 19997; depositing Lab: Fuyuhiko Tamanoi) and a GFP control plasmid pEGFPN1 (Clontech, 6085–1) using Lipofectamine 2000 (Invitrogen, 11668–019) according to the manufacturer’s instructions. .. Immunoblot analysis Muscle tissues and U2OS cells were homogenized in Radioimmunoprecipitation assay lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% NP-40 [Sigma, I3021], 0.25% Na deoxycholate [Sigma-Aldrich, 30970], 1 mm ethylenediaminetetraacetic acid) supplemented with protease inhibitor cocktail (Sigma-Aldrich, S8820).

    Stable Transfection:

    Article Title: Acid suspends the circadian clock in hypoxia through inhibition of mTOR
    Article Snippet: .. U2OS Arntl ::dLUC lines stably expressing constitutively active RHEB ( Urano et al., 2005 ) where similarly created by seeding 200,000 cells in a 6-well dish and the following day transfecting with 0.5 ng of sequence-confirmed pcDNA3-FLAG-Rheb-N153T (gift from Fuyuhiko Tamanoi, Addgene plasmids #19997) using Lipofectamine 3000. ..

    Expressing:

    Article Title: Acid suspends the circadian clock in hypoxia through inhibition of mTOR
    Article Snippet: .. U2OS Arntl ::dLUC lines stably expressing constitutively active RHEB ( Urano et al., 2005 ) where similarly created by seeding 200,000 cells in a 6-well dish and the following day transfecting with 0.5 ng of sequence-confirmed pcDNA3-FLAG-Rheb-N153T (gift from Fuyuhiko Tamanoi, Addgene plasmids #19997) using Lipofectamine 3000. ..

    Sequencing:

    Article Title: Acid suspends the circadian clock in hypoxia through inhibition of mTOR
    Article Snippet: .. U2OS Arntl ::dLUC lines stably expressing constitutively active RHEB ( Urano et al., 2005 ) where similarly created by seeding 200,000 cells in a 6-well dish and the following day transfecting with 0.5 ng of sequence-confirmed pcDNA3-FLAG-Rheb-N153T (gift from Fuyuhiko Tamanoi, Addgene plasmids #19997) using Lipofectamine 3000. ..

    Over Expression:

    Article Title: Mitochondria are required for pro‐ageing features of the senescent phenotype
    Article Snippet: .. For the overexpression of activated Rheb and PGC‐1β, MEFs were transfected with an empty vector and pcDNA3‐flag‐Rheb‐N153T (Addgene #19997) or pcDNAf:PGC‐1β (Addgene #1031) respectively, using Lipofectamine TM 2000 (Invitrogen, 11668‐019) following the manufacturer's instructions. .. YFP‐Parkin retroviral production: phoenix amphotropic cells were incubated for 24 h in antibiotic‐free medium before proceeding to transfection with a LZRS or LZRS‐YFP_Parkin vector using Lipofectamine TM 2000 (Invitrogen, 11668‐019) following the manufacturer instructions.

    Pyrolysis Gas Chromatography:

    Article Title: Mitochondria are required for pro‐ageing features of the senescent phenotype
    Article Snippet: .. For the overexpression of activated Rheb and PGC‐1β, MEFs were transfected with an empty vector and pcDNA3‐flag‐Rheb‐N153T (Addgene #19997) or pcDNAf:PGC‐1β (Addgene #1031) respectively, using Lipofectamine TM 2000 (Invitrogen, 11668‐019) following the manufacturer's instructions. .. YFP‐Parkin retroviral production: phoenix amphotropic cells were incubated for 24 h in antibiotic‐free medium before proceeding to transfection with a LZRS or LZRS‐YFP_Parkin vector using Lipofectamine TM 2000 (Invitrogen, 11668‐019) following the manufacturer instructions.



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    Figure 6. NKCC1 Independently Regulates LAT1 or the Akt (or Erk) Pathways to Control mTORC1 Activation (A) Increased LAT1 activity in NKCC1-depleted cells enhances mTORC1 activation independently of Akt or Erk. Scrambled or NKCC1-KD HeLa cells were transfected with constitutively active Flag- <t>Rheb(N153T),</t> treated with Akt and Erk1/2 inhibitor (Akti and Erk1/2i) under starvation conditions, and stimulated with EAA for 15 min, where indicated. mTORC1 and Erk1/2 activation was assessed using immunoblotting for S6K1 phosphorylation (p-p70), p-Akt (S473), and p-Erk1/2 (T202/Y204) antibodies, respectively. Lower panels depict controls for NKCC1, Flag-Rheb(N153T), Akt, and Erk expression, as indicated. (B) Constitutively active Rag complex enhances mTORC1 activation in NKCC1-KD cells in the absence of amino acids. Scrambled or NKCC1-KD HeLa cells were transfected with activated Rag complex (RagA(Q66L)/RagC(S75L)), and mTORC1 activation (p-p70/p-70 ratio) was analyzed in serum-starved cells in the absence of amino acids (i.e., in the absence of LAT1 contribution). For both (A) and (B), quantifications of activated mTORC1 (p-p70/p-70), or Akt (p-Akt(S473)/Akt), are shown beneath or beside the immunoblots and depict mean ± SEM (N = 3); p values were calculated using Student’s t tests. (C) Model: NKCC1-mediatedregulationof mTORC1 activation. NKCC1 suppresses mTORC1 activation by inhibiting the Leu transporter LAT1, the IR/PI3K/ Akt pathway, and the Erk pathway. Several com- ponents ofthe mTORC1 regulatorymachinery atthe lysosomal membrane are not included here for simplicity. See also Figure S6.
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    Figure 6. NKCC1 Independently Regulates LAT1 or the Akt (or Erk) Pathways to Control mTORC1 Activation (A) Increased LAT1 activity in NKCC1-depleted cells enhances mTORC1 activation independently of Akt or Erk. Scrambled or NKCC1-KD HeLa cells were transfected with constitutively active Flag- <t>Rheb(N153T),</t> treated with Akt and Erk1/2 inhibitor (Akti and Erk1/2i) under starvation conditions, and stimulated with EAA for 15 min, where indicated. mTORC1 and Erk1/2 activation was assessed using immunoblotting for S6K1 phosphorylation (p-p70), p-Akt (S473), and p-Erk1/2 (T202/Y204) antibodies, respectively. Lower panels depict controls for NKCC1, Flag-Rheb(N153T), Akt, and Erk expression, as indicated. (B) Constitutively active Rag complex enhances mTORC1 activation in NKCC1-KD cells in the absence of amino acids. Scrambled or NKCC1-KD HeLa cells were transfected with activated Rag complex (RagA(Q66L)/RagC(S75L)), and mTORC1 activation (p-p70/p-70 ratio) was analyzed in serum-starved cells in the absence of amino acids (i.e., in the absence of LAT1 contribution). For both (A) and (B), quantifications of activated mTORC1 (p-p70/p-70), or Akt (p-Akt(S473)/Akt), are shown beneath or beside the immunoblots and depict mean ± SEM (N = 3); p values were calculated using Student’s t tests. (C) Model: NKCC1-mediatedregulationof mTORC1 activation. NKCC1 suppresses mTORC1 activation by inhibiting the Leu transporter LAT1, the IR/PI3K/ Akt pathway, and the Erk pathway. Several com- ponents ofthe mTORC1 regulatorymachinery atthe lysosomal membrane are not included here for simplicity. See also Figure S6.
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    Figure 6. NKCC1 Independently Regulates LAT1 or the Akt (or Erk) Pathways to Control mTORC1 Activation (A) Increased LAT1 activity in NKCC1-depleted cells enhances mTORC1 activation independently of Akt or Erk. Scrambled or NKCC1-KD HeLa cells were transfected with constitutively active Flag- <t>Rheb(N153T),</t> treated with Akt and Erk1/2 inhibitor (Akti and Erk1/2i) under starvation conditions, and stimulated with EAA for 15 min, where indicated. mTORC1 and Erk1/2 activation was assessed using immunoblotting for S6K1 phosphorylation (p-p70), p-Akt (S473), and p-Erk1/2 (T202/Y204) antibodies, respectively. Lower panels depict controls for NKCC1, Flag-Rheb(N153T), Akt, and Erk expression, as indicated. (B) Constitutively active Rag complex enhances mTORC1 activation in NKCC1-KD cells in the absence of amino acids. Scrambled or NKCC1-KD HeLa cells were transfected with activated Rag complex (RagA(Q66L)/RagC(S75L)), and mTORC1 activation (p-p70/p-70 ratio) was analyzed in serum-starved cells in the absence of amino acids (i.e., in the absence of LAT1 contribution). For both (A) and (B), quantifications of activated mTORC1 (p-p70/p-70), or Akt (p-Akt(S473)/Akt), are shown beneath or beside the immunoblots and depict mean ± SEM (N = 3); p values were calculated using Student’s t tests. (C) Model: NKCC1-mediatedregulationof mTORC1 activation. NKCC1 suppresses mTORC1 activation by inhibiting the Leu transporter LAT1, the IR/PI3K/ Akt pathway, and the Erk pathway. Several com- ponents ofthe mTORC1 regulatorymachinery atthe lysosomal membrane are not included here for simplicity. See also Figure S6.
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    Figure 6. NKCC1 Independently Regulates LAT1 or the Akt (or Erk) Pathways to Control mTORC1 Activation (A) Increased LAT1 activity in NKCC1-depleted cells enhances mTORC1 activation independently of Akt or Erk. Scrambled or NKCC1-KD HeLa cells were transfected with constitutively active Flag- Rheb(N153T), treated with Akt and Erk1/2 inhibitor (Akti and Erk1/2i) under starvation conditions, and stimulated with EAA for 15 min, where indicated. mTORC1 and Erk1/2 activation was assessed using immunoblotting for S6K1 phosphorylation (p-p70), p-Akt (S473), and p-Erk1/2 (T202/Y204) antibodies, respectively. Lower panels depict controls for NKCC1, Flag-Rheb(N153T), Akt, and Erk expression, as indicated. (B) Constitutively active Rag complex enhances mTORC1 activation in NKCC1-KD cells in the absence of amino acids. Scrambled or NKCC1-KD HeLa cells were transfected with activated Rag complex (RagA(Q66L)/RagC(S75L)), and mTORC1 activation (p-p70/p-70 ratio) was analyzed in serum-starved cells in the absence of amino acids (i.e., in the absence of LAT1 contribution). For both (A) and (B), quantifications of activated mTORC1 (p-p70/p-70), or Akt (p-Akt(S473)/Akt), are shown beneath or beside the immunoblots and depict mean ± SEM (N = 3); p values were calculated using Student’s t tests. (C) Model: NKCC1-mediatedregulationof mTORC1 activation. NKCC1 suppresses mTORC1 activation by inhibiting the Leu transporter LAT1, the IR/PI3K/ Akt pathway, and the Erk pathway. Several com- ponents ofthe mTORC1 regulatorymachinery atthe lysosomal membrane are not included here for simplicity. See also Figure S6.

    Journal: Cell reports

    Article Title: The Ion Transporter NKCC1 Links Cell Volume to Cell Mass Regulation by Suppressing mTORC1.

    doi: 10.1016/j.celrep.2019.04.034

    Figure Lengend Snippet: Figure 6. NKCC1 Independently Regulates LAT1 or the Akt (or Erk) Pathways to Control mTORC1 Activation (A) Increased LAT1 activity in NKCC1-depleted cells enhances mTORC1 activation independently of Akt or Erk. Scrambled or NKCC1-KD HeLa cells were transfected with constitutively active Flag- Rheb(N153T), treated with Akt and Erk1/2 inhibitor (Akti and Erk1/2i) under starvation conditions, and stimulated with EAA for 15 min, where indicated. mTORC1 and Erk1/2 activation was assessed using immunoblotting for S6K1 phosphorylation (p-p70), p-Akt (S473), and p-Erk1/2 (T202/Y204) antibodies, respectively. Lower panels depict controls for NKCC1, Flag-Rheb(N153T), Akt, and Erk expression, as indicated. (B) Constitutively active Rag complex enhances mTORC1 activation in NKCC1-KD cells in the absence of amino acids. Scrambled or NKCC1-KD HeLa cells were transfected with activated Rag complex (RagA(Q66L)/RagC(S75L)), and mTORC1 activation (p-p70/p-70 ratio) was analyzed in serum-starved cells in the absence of amino acids (i.e., in the absence of LAT1 contribution). For both (A) and (B), quantifications of activated mTORC1 (p-p70/p-70), or Akt (p-Akt(S473)/Akt), are shown beneath or beside the immunoblots and depict mean ± SEM (N = 3); p values were calculated using Student’s t tests. (C) Model: NKCC1-mediatedregulationof mTORC1 activation. NKCC1 suppresses mTORC1 activation by inhibiting the Leu transporter LAT1, the IR/PI3K/ Akt pathway, and the Erk pathway. Several com- ponents ofthe mTORC1 regulatorymachinery atthe lysosomal membrane are not included here for simplicity. See also Figure S6.

    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-b-actin Sigma-Aldrich Cat# A2228; RRID:AB_47669 Mouse anti-FLAG Sigma-Aldrich Cat# F1804; RRID:AB_262044 Mouse anti-haemagglutinin (HA) Covance Cat# MMS-101R; RRID:AB_10063630 Mouse anti-V5 AbD Serotec Cat# MCA1360; RRID:AB_322378 Rabbit anti-NKCC1 Abcam Cat# ab5979; RRID:AB_944433 Rabbit anti-NKCC1 Cell Signaling Cat# D13A9; RRID:AB_10830068 Rabbit anti-phospho S6K (T389) Cell Signaling Cat# 9234; RRID:AB_2269803 Rabbit anti-4E-BP1 Cell Signaling Cat# 9452; RRID:AB_331692 Rabbit anti-phospho 4E-BP1 (T37/46) Cell Signaling Cat# 9459S; RRID:AB_330985 Rabbit anti-LAT1 Cell Signaling Cat# 5347S; RRID:AB_10695104 Rabbit anti-phospho Akt (T473) Cell Signaling Cat# 9271; RRID:AB_329825 Rabbit anti-phospho Tuberin/TSC2 (S939) Cell Signaling Cat# 3615; RRID:AB_2207796 Rabbit anti-phospho p38 MAPK (T180/Y182) Cell Signaling Cat# 9211S; N/A Mouse anti-phospho p38 MAPK (T180/Y182) BD Bioscience Cat# 612280; RRID:AB_399597 Rabbit anti- phosphor Erk (T202/Y204) Cell Signaling Cat# 4370; RRID:AB_2315112 Rabbit anti-IGF-1Rb Cell Signaling Cat# 3027; RRID:AB_2122378 Mouse anti-vinculin Santa Cruz Cat# sc-25336; RRID:AB_628438 Mouse anti-S6K Santa Cruz Cat# sc-230; RRID:AB_632156 Mouse anti-IRb Santa Cruz Cat# sc-57324;RRID:AB_784102 Mouse anti-Akt BD Biosciences Cat# 610860; RRID:AB_398179 Rabbit anti-Erk Cell Signaling Cat# 4695; RRID:AB_390779 Rabbit anti-mTOR Cell Signaling Cat# 2983; RRID:AB_2105622 Mouse anti-LAMP1 Abcam Cat# ab25630; RRID:AB_470708 Rabbit anti-p38 MAPK Cell Signaling Cat# 9212; AB_330713 Mouse anti-GFP Abcam Cat# ab1218; RRID:AB_298911 Goat anti-mouse IgG, Alexa Fluor 488 Thermo Fisher Cat# A11029; RRID:AB_2534088 Goat anti-rabbit IgG, Alexa Fluor 555 Thermo Fisher Cat# A21429; RRID:AB_2535850 Goat anti-rabbit IgG, Alexa Fluor 647 Thermo Fisher Cat# A21245; RRID:AB_2535813 Succinimidyl ester conjugated to Alexa Fluor 647 Life Technologies Cat# A20006; RRID: N/A Bacterial and Virus Strains E. coli DH5a Thermo Fisher Cat# 18258012 Chemicals, Peptides, and Recombinant Proteins Rheb (N153T) Addgene Cat# 19997 Rag C (S75L) Addgene Cat# 19305 Rag A (Q66L) Gift from Dr. T Meyer N/A Tetracycline hydrochloride Sigma-Aldrich Cat# T7660 Anti-FLAG M2 affinity agarose Sigma-Aldrich Cat# A2220 Humulin R Lilly Cat# U-100 IGF-1 Sigma-Aldrich Cat# I13769 BMS-536924 Tocris Cat# 4774 Bumetanide Sigma-Aldrich Cat# B3023 PD98059 Cell Signaling Cat# 9900 (Continued on next page) Cell Reports 27, 1886–1896.e1–e6, May 7, 2019 e1

    Techniques: Control, Activation Assay, Activity Assay, Transfection, Western Blot, Phospho-proteomics, Expressing, Membrane