Immunoblotting was performed as previously described [14 (link),16 (link)]. Cell lysates were prepared for immunoblotting, using antibodies against NEDD8, NAE1, UBA3, UBC12, WEE1, cullin-1(Santa Cruz Biotechnology), ORC1, p27, cleaved Caspase 3, cleaved PARP, CDT1, H2AX, p-H2AX, CHK1, p-CHK1, GAPDH, p-Histone H3 (Cell Signaling), IκB-α, p-IκB-α, p21 (Epitomics, Inc.).
P iκbα
Manufactured by Abcam
Sourced in United States, United Kingdom
P-IκBα is a protein that plays a key role in the regulation of the NF-κB signaling pathway. It is the phosphorylated form of the IκBα protein, which acts as an inhibitor of NF-κB transcription factors. P-IκBα is a crucial component in the activation and nuclear translocation of NF-κB, making it a valuable tool for researchers studying this important cellular signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (14 mentions)
Gapdh, by Abcam (6 mentions)
P ikkα β, by Abcam (5 mentions)
Cleaved caspase 3, by Abcam (5 mentions)
Nf κb p65, by Abcam (5 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
P ikkβ, by Abcam (4 mentions)
Bcl 2, by Abcam (4 mentions)
Vimentin, by Abcam (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
P erk1 2, by Abcam (3 mentions)
P jnk, by Abcam (3 mentions)
P nf κb, by Abcam (3 mentions)
P ikkα, by Abcam (3 mentions)
P akt, by Abcam (3 mentions)
P pi3k, by Abcam (3 mentions)
Caspase 3, by Abcam (3 mentions)
Nf κb, by Abcam (3 mentions)
Hmgb1, by Abcam (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
50 protocols using p iκbα
1
Immunoblotting Analysis of Cell Signaling
2
Immunoblotting Analysis of Cell Signaling
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Cell lysates were prepared for immunoblotting using antibodies against Wee1, cullin-1, NF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ORC1, P27, cleaved Caspase 3, cleaved PARP, CDT1, H2AX, p-H2AX, CHK1, p-CHK1, Bcl-2,Bcl-xL, Mcl-1, c-IAP1, c-IAP2, Survivin, XIAP, BAX, Bik, Bim, BID, Bak, ROC1 (Cell Signaling Technology, CST, Danvers, MA, USA), IκB-α, p-IκB-α, P21, RhoA, Cdc42 (Epitomics, Burlingame, CA, USA), NOXA (CALBIOCHEM, San Diego, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma).
3
Western Blot Analysis of Signaling Pathways
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hDPCs were lysed using RIPA lysis buffer. Total protein was measured using a BCA Protein Assay Kit (Beyotime, Haimen, China). Thirty micrograms of protein was separated by electrophoresis on 8% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) with transfer buffer containing 10% methanol. After blocking with TBST containing 5% nonfat milk at room temperature for 1 h, the membranes were probed overnight at 4°C with the primary antibodies (1:2000) including IKKα/β, p-IKKα/β, IκBα, p-IκBα, p65, p-p65, p38, p-p38, ERK1/2, p-ERK1/2, JNK, p-JNK (CST, USA) and GAPDH (Abcam, UK). Subsequently, the membranes were incubated for 1 h with secondary antibodies at a dilution of 1:2000 (CST, USA) at room temperature. After the membranes were thoroughly washed with TBST buffer, bands with target proteins were visualized with enhanced chemiluminescence reagents (Millipore, USA) and observed using an ImageQuant LAS 4000 mini system (GE Healthcare Life Sciences, USA). The blots were quantified and normalized using the ImageJ 1.47 software program (National Institutes of Health, MD, USA).
4
Comprehensive Hepatoprotective Mechanism Study
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DHM (purity ≥99.0%) and TAA (purity ≥99.0%) were purchased from Sigma. Hematoxylin-eosin (H and E), TUNEL and Masson stain kits, and commercial assay kits for ALT and AST, SOD, GSH, MDA were obtained from the Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The immunofluorescent staining kits and the enhanced chemiluminescence (ECL) kit were purchased from Beyotime science and technology Co., Ltd. (Beijing, China). The antibody of rabbit monoclonal α-SMA, TGF-β1, CYP2E1, Cleaved Caspase-3, Caspase3, Cleaved Caspase-9, Caspase-9, IκB kinase α (IKK-α), IκB kinase β (IKK-β), p-IKKα, p-IKKβ, inhibitor of IκBα (IκBα), p-IκBα, NF-κB, p-NF-κB, PI3K, p-PI3K, Akt, p-Akt, B-associated X (Bax), Bcl-2, β-actin and secondary antibodies for western blot were all obtained from Abcam (Cambridge, United Kingdom).
5
Molecular Profiling of Cancer Signaling
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RNA extraction, RT-qPCR, western blotting, and transwell assay were performed as described previously [18 (link)]. The following antibodies were used for western blot analysis: GAPDH (CST), p65 (CST), p-p65 (CST), IκBα (CST), p-IκBα (CST), MMP14 (CST), histone 3 (Abcam), and tubulin (Abcam). The primers used for PCR and the sequences of the shRNAs used in the study are shown in Table S1 .
6
Immunodetection of Key Signaling Proteins
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The following antibodies were used: RNF8 (Proteintech, # 14112-1-AP), β-tubulin (Affinity, # T0023), CD3 (Affinity, # DF6594), IL-12 (Affinity, # AF5133), IFN-γ (Affinity, # DF6045), CXCL-9 (Affinity, # DF9920), CXCL-10 (Affinity, # DF6417), galectin-3 (Proteintech, # 14979-1-AP), HIF-1α (Proteintech, # 66730-1-Ig), p-IκBα (Abcam, # ab133462), NF-κB (Abcam, # ab16502), P-NF-κB (Santacruze, # sc-101752), PCNA (Proteintech, # 60097-1-Ig), Flag (Proteintech, # 20543-1-AP), HA (Proteintech, # 66006-2-Ig), His (Proteintech, # 66005-1-Ig), ubiquitin (CST, # 91112 S), K48 (CST, # 12805), K63 (CST, # 5621S). Secondary anti-mouse (# SA00001-1), and anti-rabbit (# SA00001-2) horseradish-coupled antibodies were from Proteintech.
7
Anti-inflammatory Effects of 4GMV Cream
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4GMV (Batch No. 111523‐201610, purity >96.10%) was obtained from Chinese Food and Drug Inspection Institute (China) and IMQ cream from Sichuan Mingxin Pharmaceutical Co. (China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Corning (USA). LPS (from E. coli, isotype 055: B5) was obtained from Sigma Chemical Co. (USA) and PBS from Solarbio (China). Cell counting kit (CCK)-8 was purchased from Dojindo (Japan), nitric oxide assay kit from Applygen Co. (China), and prostaglandin E2 (PGE2) assay kit from Tianjin Createch Biotechnology Co. (China). Total RNA Extraction kit and One-step RT-PCR kit were obtained from Invitrogen (USA). Antibodies for TLR4, p-ERK, p-IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), and p-p65 were purchased from Cell Signaling Technology (USA) and antibodies for β-actin, cyclo-oxigenase-2 (COX-2), inducible nitric oxide synthase (iNOS), TLR4, p38, p-p38, extracellular signal-regulated kinase (ERK), p-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p-IκBα, and p-p65 were purchased from Abcam (UK). Methotrexate (MTX) was obtained from Shanghai Pharmaceutical Co. (China) and Vaseline from Lanlianfeitian Petrochemical Co. (China).
8
Ginsenoside Rh2 Neuroprotective Mechanisms
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Shanghai YuanYe Biotechnology Co., Ltd. (Shanghai, China) supplied 20(S)-ginsenoside Rh2 (purity ≥ 98%). Dalian Meilun Biological Technology Co., Ltd. (Dalian, China) provided SPI which served as a control drug. The sodium carboxymethyl cellulose (0.5% w/v) (Sangon Biotech Co., Ltd., Shanghai, China) was used to suspend GRh2 or SPI. Nissl staining solution was supplied by Xi'an Hat Biotechnology Co., Ltd (Xi'an, China). Primary antibodies used in immunohistochemical staining against tyrosine hydroxylase (TH), HMGB1, NeuN, Annexin V, ionized calcium-binding adapter molecule-1 (Iba-1), TLR4 and phospho-NF-κB p65 (p-NF-κB p65) were purchased from Abcam Inc. (Cambridge, MA, USA). The antibodies used for Western blot against TLR4, indoleamine 2,3-dioxygenase (IDO), NF-κB p65, p-NF-κB p65, myeloid differentiation primary response gene 88 (MyD88), tumor necrosis factor-alpha (TNF-α), α-tubulin and β-actin were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA), antibodies against Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), TH, HMGB1, interferon-gamma (IFN-γ), Iba-1, inducible nitric oxide synthase (iNOS), NF-κB inhibitor-alpha (IκB-α) and p-IκB-α were procured from Abcam (Cambridge, MA, USA).
9
Protein Extraction and Western Blot Analysis
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Total protein was extracted with RIPA buffer (Thermo Fisher Scientific). Protein
concentration was detected using the Easy II Protein Quantitative Kit (BCA) (TransGen
Biotech, Beijing, China). Then, the proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride
membranes (Millipore, Billerica, MA, USA). The membranes were incubated at 4°C
overnight with primary antibodies against Bax (Abcam, Cambridge, UK), B-cell lymphoma
2 (Bcl-2; Abcam), cleaved caspase 3 (Abcam), Beclin-1 (Abcam), microtubule-associated
protein 1 light chain 3 (LC3)-I (Abcam), LC3-II (Abcam), p-IκBα
(Abcam), IκBα (Abcam), p-p65 (Abcam), p65 (Abcam), or GAPDH (Abcam) at
a dilution ratio of 1:1,000. Next, the membranes were incubated with the
corresponding horseradish peroxidase–conjugated secondary antibody (1:4,000;
Abcam) for 2 h at room temperature. The protein bands were visualized by
enhanced chemiluminescence reagents (Millipore).
concentration was detected using the Easy II Protein Quantitative Kit (BCA) (TransGen
Biotech, Beijing, China). Then, the proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride
membranes (Millipore, Billerica, MA, USA). The membranes were incubated at 4°C
overnight with primary antibodies against Bax (Abcam, Cambridge, UK), B-cell lymphoma
2 (Bcl-2; Abcam), cleaved caspase 3 (Abcam), Beclin-1 (Abcam), microtubule-associated
protein 1 light chain 3 (LC3)-I (Abcam), LC3-II (Abcam), p-IκBα
(Abcam), IκBα (Abcam), p-p65 (Abcam), p65 (Abcam), or GAPDH (Abcam) at
a dilution ratio of 1:1,000. Next, the membranes were incubated with the
corresponding horseradish peroxidase–conjugated secondary antibody (1:4,000;
Abcam) for 2 h at room temperature. The protein bands were visualized by
enhanced chemiluminescence reagents (Millipore).
10
Immunoblot and Immunohistochemistry for Protein Expression
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Detection of protein expression by immunoblot was carried out according to the established protocols described previously [40 (link)]. JMJD3 (Cell Signal, Danvers, MA) (Cat# 3457), IRF4 (Abcam, Cambridge, MA) (Cat# ab5184- 1), caspase-3 (Cell Signal) (Cat# 9665), Bcl-2 (Cell Signaling), (Cat# 2870), NF-κB (P65 subunit) (Abcam) (Cat# ab32536), NF-κB (P105/50 subunit) (Abcam) (Cat# ab32360), laminA (Cat# ab8980), (Abcam), IκBα (1:1000, Cat# ab32518, Abcam), p-IκBα (1:1000, Cat# ab133462, Abcam) and β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (Cat# sc-47778) antibodies were used. Secondary horseradish peroxidase - conjugated goat anti-rabbit or mouse antibodies (Bio-Rad) were detected by the enhanced chemiluminescence reagent (Millipore, Billerica, MA). Paraffin sections were incubated at 4°C overnight with the primary antibody after dewaxing and hydration. Anti-JMJD3 (Abcam) (Cat# ab38113) or -IRF4 (Abcam) were used at 1:50 dilution. Slides were incubated with a biotinylated secondary antibody for 1.5 h, then developed with avidin-peroxidase and DAB and counterstained with hematoxylin. Dehydrated slides were mounted with neutral resin.
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