The antibodies to CA9, p-RelA, RelA, p-IKKβ, IKKβ, p-IκBα, IκBα, LDH, HIF1α, SQSTM1, cleaved-caspase-3, cleaved-PARP1, NRF2, and actin were obtained from Cell Signaling Technology. The antibodies to GPX4 and CA9 were obtained from Abcam. The antibodies to LC3, MLKL, RIPK3, OPRL1, OPRM1, and HMGB1 were obtained from NOVUS. Desferrioxamine, β-mercaptoethanol, N-acetyl-l-cysteine, N-Acetyl-l-alaine, hydrogen peroxide solution, gemcitabine hydrochloride, sulfasalazine, cobalt chloride, cycloheximide, tocopherol, necrosulfonamide, hydroxychloroquine sulfate, EDTA, cytochalasin B, cytochalasin D, paclitaxel, crystal violet, protease inhibitor cocktail, Z-VAD-FMK, TNF, staurosporine, cycloheximide, and lipopolysaccharides were obtained from Sigma-Aldrich. Erastin, ferrostatin-1, lapatinib, JTC801, and compound libraries were obtained from Selleck Chemicals. BANORL24, SB612111, UFP101, and Trap101 were obtained from Tocris Bioscience.
P rela
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
P-RelA is a primary antibody that specifically recognizes the phosphorylated form of the NF-κB subunit RelA (also known as p65). It can be used to detect the activated form of RelA in cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
P iκbα, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Tocopherol, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Hydrogen peroxide solution, by Merck Group (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Necrosulfonamide, by Merck Group (1 mentions)
Gemcitabine hydrochloride, by Merck Group (1 mentions)
Sb 612111, by Bio-Techne (1 mentions)
Cleaved parp 1, by Cell Signaling Technology (1 mentions)
Cobalt chloride, by Merck Group (1 mentions)
Hydroxychloroquine sulfate, by Merck Group (1 mentions)
Tumor necrosis factor (tnf), by Merck Group (1 mentions)
7 protocols using p rela
1
Comprehensive Immunoblot Analysis
2
Western Blot Analysis of Apoptosis Regulators
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Proteins were extracted with CelLyticTM M Cell Lysis Reagent (Sigma‐Aldrich, Dorset, UK) supplied with protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, Dorset, UK). Proteins were mixed with NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min before analysis by western blot analysis. Proteins were subjected to 4–20% NuPAGE gels (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Sigma‐Aldrich, Dorset, UK) at 20 V for 1 hr by semidry transfer. PVDF membrane was blocked with 5% nonfat milk in TBST for 1 hr and then incubated with primary antibodies overnight at 4°C against the following targets: Bcl‐2 (100), RelA (NF‐κB p65, F‐6), Bcl‐XL (H‐5), Bax (2D2), Survivin (D‐8), and LDH (H‐10) (Santa Cruz Biotechnology, Inc., Dallas, TX); β‐actin (AC‐74) (Sigma‐Aldrich, Dorset, UK); p‐RelA (phospho‐NF‐κB p65‐Ser536), p‐STAT3 (phosphor‐STAT3‐Tyr705), p‐AKT, AKT, Mcl‐1, and Rb antibodies (Cell Signaling Technology‐New England Biolabs, Hitchin, UK). Bound antibodies were detected using appropriate horseshoeradish peroxidase‐conjugated secondary antibodies and visualized by GeneSnap (SynGene, Cambridge, UK) after adding ECL plus (GE Healthcare Life Science, Hatfield, UK).
3
SiHa Cell Line Culture and Transfection
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The human cervical cancer cell line SiHa was cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS, 100 mg/ml streptomycin, 100 units/ml penicillin at 37°C with 5% CO2. pCMV–E2 (E2 expression plasmid containing cytomegalovirus promoter) (Dr Lawrence Banks, ICGEB, Italy) and pGL4.32–NF-κB–Luc (Promega) plasmids were used. SiHa cells were transfected using Lipofectamine 2000 (Invitrogen) as per manufacturer's protocol. Antibodies to phospho-RelA (p-RelA) (phosphorylated Rel-like domain-containing protein A), p21, Bcl2, p27Kip1 (cyclin-dependent kinase inhibitor p27) and high-mobility group protein A (HMGA)1 (93H1, 2941, 2774, D69C12 and D6A4 respectively, from Cell Signaling), RelA, HPV16 E2, p53 and HMG-1 (9E10, TVG261, 126 and W-18, respectively, from Santa Cruz), p16INK4a (cyclin-dependent kinase inhibitor 2A) (1963, Epitomics), β-actin (A2228, Sigma–Aldrich) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were procured. Human recombinant-TNF-α (PeproTech), resazurin (Himedia) and PI (propidium iodide) (Invitrogen) were also used in the present study.
4
Western Blot Analysis of TA Muscle
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TA muscles were dissected, minced, and homogenized in lysis buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 Mm NaCl, 1% TRITON) supplemented with protease and phosphatase inhibitors (complete Mini EDTA-free and PhosSTOP, ROCHE). Proteins (30-50 micrograms) were separated by SDS-PAGE and transferred to PVDF membrane (Invitrogen). Unspecific binding was blocked in 5% not fat dry milk in TBST buffer (20 mM Tris HCl pH 7.6, 137 mM NaCl, 0.5% Tween 20). The membranes were incubated overnight, with the appropriate primary antibody diluted in 5% BSA (Sigma) in TBST. After washing in TBST, membranes were incubated with secondary antibodies HRP-conjugate (Bio-Rad 170-6515 or 170-6516) and signals were detected by using ECL chemistry (Advansta). Images were aquired using films or ChemiDoc MP imaging system (Bio-Rad). Densitometric analyses were performed by measuring band intensity for each sample using Image J software. The following primary antibodies were used: Gapdh (Santa Cruz sc-32233), Cybb (BD bioscience 611414), P-Rela (Cell Signalling 3033), TOT-Rela (Cell Signalling 4764), 4-HNE (Abcam 46545)
5
Immunoblotting Analysis of Cellular Signaling
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Cells were lysed in lysis buffer (150 mM NaCl, 30 mM TRIS, 0.1% Triton X-100, 10% glycerol and 1X protease inhibitor cocktail). Protein concentration was quantified using the BCA method (Pierce). 50 μg of total protein was resolved either on 4–12% NuPAGE® Bis-Tris or 3–8% Tris-Acetate mini-gels (Invitrogen) and transferred to PVDF membrane. Membranes were blocked for 1 h at room temperature (RT) either with 5% non-fat dry milk or 5% Bovine serum albumin (BSA) in Tris-buffered saline containing 0.01% Tween 20 (TBS-T) and incubated overnight at 4 °C with primary antibodies. Chemiluminescence was detected using Novex® ECL substrate reagent and High performance film (GE Healthcare). β-actin, α-tubulin or GAPDH were used as loading controls. Antibodies for PDGFR-α (#5241), PDGFR-β (-#3169), GRB2 (#3972), pERKs (#9101), RAS (#8955), pAKT (#4060), totAKT (#2966), AKT2(#5239), pp53 (Ser15) (#9284), pRelA (#3033), totRelA (#6956), IKKβ (#8943), GAPDH (#3683) were purchased from Cell Signaling Technology. SOS1 (ab140621), IQGAP1 (ab86064), ERK2 (ab124362), p21 (ab109520), CDK1 (ab19), p53 (ab179477) antibodies were obtained from Abcam. MDM2 antibody was purchased from Calbiochem (OP115). RALA (sc-374462), Raf-1(sc-227) and β-actin (SC-47778) antibodies were purchased from Santa Cruz Biotechnology.
6
Western Blot Analysis of Signaling Proteins
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Cell lysates were prepared in radioimmunoprecipitation assay lysis buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Pierce, Waltham, MA, USA). Protein concentration was measured by the BCA Protein Assay kit (Pierce). Equal amount of protein (30 μg) was separated by NuPAGE Novex Bis-Tris 4–12% gels (Life Technologies) and transferred onto a nitrocellulose membrane using iBlot Gel Transfer Device (Life Technologies). The membrane was incubated with primary antibodies for 2 h at room temperature. Antibodies against p-AKT, p-ERK1/2, p-IκBα, p-RELA, p-S6, total AKT, ERK1/2 and GAB2 were purchased from Cell Signaling Technology. After incubation with the appropriate horseradish peroxidase linked secondary antibodies (Bio-Rad, Hercules, CA, USA) for 2 h at room temperature, the membrane was incubated with Enhanced Chemiluminescence Plus substrate (Pierce) and signals were detected by Pierce CL-Xposure Film. Expression of β-actin was assessed as an internal loading control by use of a specific antibody (sc-8432-HRP; Santa Cruz Biotechnology, Dallas, TX, USA). The intensity of bands was quantified by using Fiji image processing software (http://fiji.sc/Fiji ).
7
Immunoblotting of NF-κB Pathway
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Antibodies against p21 and HIF-1α were from BD Biosciences; p-RelA, pIKK, pIκB-α, IKK, IκB-α and β-actin were from Cell Signaling; IL-8 from R&D systems; CDK6 and RelA from Santa Cruz Biotechnology. Antibodies against Relish, Dorsal, Cactus, α-Tubulin and Tango were from Developmental Studies Hybridoma Bank (DSHB). Enhanced chemiluminescence (Pierce) was used for detection.
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