Cells plated in media containing 5% FCS were treated with solvent (0.1% DMSO) in the absence or presence of various concentrations of FASN-inhibitor for 1 to 72 h. After lysis, proteins were subjected to SDS–PAGE, blotted and immunostained as described [45 (link), 46 (link)] using anti-FASN (BD Biosciences, San Jose, CA; 1:500), anti-AKT, anti-pAKT, anti-AMPKα, anti-pAMPKα, anti-DEPTOR, anti-ERK, anti-pERK, anti-HIF-1α, anti-mTOR, anti-pmTOR, anti-p70S6K, anti-pp70S6K, anti-S6, anti-pS6, anti-4EBP1, anti-p4EBP1, anti-cleaved caspase 3, anti-PARP1 (Cell Signaling Technology, Boston, MA; 1 : 500–1:3 000), anti-REDD1 (Proteintech, Manchester, UK; 1:1 000), and anti-actin (Santa Cruz Biotechnology, Dallas, TX; 1:1 000). Secondary antibodies were peroxidase-labelled donkey-anti-rabbit (Promega, Madison, WI) or donkey-anti-goat IgG (Santa Cruz Biotechnology) at 1:15 000, or chicken-anti-mouse IgG (Santa Cruz Biotechnology) at 1:10 000. Detection was by enhanced chemiluminescence.
Anti redd1
Manufactured by Proteintech
Sourced in Jamaica
Anti-REDD1 is a laboratory reagent used for the detection and quantification of REDD1 (Regulated in Development and DNA Damage responses 1) protein. REDD1 is a protein that plays a role in the regulation of cellular processes. The Anti-REDD1 product provides a tool for researchers to study the expression and function of REDD1 in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Santa Cruz Biotechnology (2 mentions)
P70s6k1 t389, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Fortis Life Sciences (2 mentions)
Precast criterion gels, by Bio-Rad (2 mentions)
Ecl plus reagent, by Bio-Rad (2 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti p p70s6k, by Cell Signaling Technology (1 mentions)
Anti akt, by BD (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti 4e bp1, by Cell Signaling Technology (1 mentions)
Anti ampkα, by Cell Signaling Technology (1 mentions)
Anti fasn, by BD (1 mentions)
Donkey anti goat igg, by Santa Cruz Biotechnology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti p 4ebp1, by Cell Signaling Technology (1 mentions)
Anti parp1, by Cell Signaling Technology (1 mentions)
Anti s6, by Cell Signaling Technology (1 mentions)
Anti ps6, by Cell Signaling Technology (1 mentions)
6 protocols using anti redd1
1
Immunoblot Analysis of FASN Inhibitor Effects
2
Western Blot Analysis of REDD1 and p70S6K1
3
Western Blot Analysis of Signaling Pathways
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HUVECs and HLECs were suspended in RIPA buffer [50 mΜ Tris-HCl (pH 8.0), 150 mΜ NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS)] and incubated on ice for 30 min for complete cell lysis. After centrifugation at 12,000 × g for 10 min, lysates (25 μg of protein) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, followed by Western blotting with antibodies against target proteins. Antibodies against mTOR (#2972), p-mTOR (#2971), S6K (#9202), p-S6K (#9205), 4E-BP1 (#9644), p-4E-BP1 (#2855), ERK (#9102), p-ERK (#9106), Akt (#9272), p-Akt (#9271), VEGFR-1 (#2893), VEGFR-2 (#2479), p-VEGFR-2 (#2478), EGFR (#4267), and IGF-1Rβ (#3027) were purchased from Cell Signaling Technology (Denver, CO). Other antibodies used included anti-β-actin (#A5441; Sigma-Aldrich, Burlington, MA), anti-REDD1 (#10638-1-AP; Proteintech, Rosement, IL), anti-VEGFR-3 (#AF349; R&D Systems), anti-p-VEGFR-3 (#CY111; Cell Applications, Inc., San Diego, CA), and goat anti-mouse IgG (#31430; Invitrogen), goat anti-rabbit IgG (#31460; Invitrogen), and rabbit anti-goat IgG (#31402; Invitrogen) secondary antibodies. Relative protein levels were quantified using ImageJ.
4
Western Blot Analysis of Protein Targets
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Cells were treated as described above, washed once in phosphate-buffered saline, lysed and harvested in Laemmli sample buffer, and boiled for 5 min. An equal volume of each sample was fractionated by SDS-PAGE using Bio-Rad precast Criterion gels (Bio-Rad, Hercules, CA). Fractionated samples were transferred onto PVDF membranes and blocked for 1 h with 5% nonfat dry milk (NFDM) in Tris-buffered saline with 0.1% Tween20 (TBST). Membranes were incubated overnight at 4 °C with one of the following primary antibodies: anti-REDD1 (ProteinTech Group Inc., Chicago, IL, Cat. # 10638), p70S6K1 T389 (Cell Signaling, Danvers, MA, Cat. # 9205), alpha-Tubulin (Santa Cruz, Dallas, TX, Cat. # sc-32293). Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibody (Bethyl, Montgomery, TX) in 5% NFDM in TBST at room temperature for one hour then washed thrice in TBST. Blots were developed with ECL Plus reagents (Bio-Rad, Hercules, CA) using a FluorChem M Multifluor System (ProteinSimple, San Jose, CA). Western blot densitometry was quantitated using Image J Software (National Institutes of Health).
5
Molecular Mechanisms of Chemotherapeutic Agents
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Paclitaxel (NSC 125 973), cisplatin (s1166), and doxorubicin (s1208) were purchased from Selleck. Actinomycin D (HY‐17559) and rapamycin (HY‐10219) were purchased from MCE. CQ (C6628) was purchased from Sigma. Antibodies used in immunoblotting: mTOR (2972S, 1:1000), p‐mTOR (Ser2448) (5536S, 1:1000), SQSTM1/p62 (88588S, 1:1000), BECN1 (D40C5, 1:1000), LC3 (3868S, 1:1000) were purchased from Cell Signaling Technologies. Anti‐REDD1 (10638‐1‐AP, 1:1000), anti‐AUF1 (12770‐1‐AP, 1:1000), anti‐ZFP36 (12737‐1‐AP, 1:1000), anti‐KHSRP (55409‐1‐AP, 1:1000), anti‐β‐actin (20536‐1‐AP,1:5000), were purchased from Proteintech. GAPDH (GB11002) was purchased from servicebio. LC3 (L7543, 1:2000) was purchased from Sigma. Ki67 (MAB‐0672) was purchased from MXB Biotechnologies. Anti‐H3K27 (ab4729, abcam), anti‐TCF4 (22337‐1‐AP, proteintech) and normal IgG (2729, CST) were used in the CHIP assay. Normal IgG/Peroxidase‐conjugated AffiniPure Goat Anti‐Rabbit/Mouse IgG (H+L) was purchased from Jackson Immuno Research.
6
Western Blot Analysis of Signaling Proteins
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Western blots were performed as previously described [35 ]. Briefly, the following antibodies were incubated on membranes overnight: anti-P-S6K1 (1/1000) (Cell Signaling Technology # 9206,) anti-Myogenin (1/100) (Santa-Cruz, sc-576), and anti-REDD1 (1/1000) (ProteinTech 10638-1-AP). The next day, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at 1/3000 for 1 h at room temperature. Membranes were developed with an enhanced chemiluminescent (ECL) reagent from Biorad using a Chemidoc™ Touch Imaging System (BioRad). The stain-free system from Biorad was used to quantify the total protein load and to normalize phosphorylated S6K1.
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