Changes in the expression levels of proteins in the mice lung tissues were examined by western blotting. Briefly, the lung tissue were homogenised in a lysis buffer (RIPA lysis buffer: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA pH 8.0 with 1 mM PMSF, 1 mM Na3VO4, and Protease Inhibitor (Cocktail-P2714, Sigma)). Protein lysate (30 ng) was subjected to SDS-PAGE gel. Then proteins were transferred from the gels to PVDF membrane. The membranes were blocked with 5% nonfat milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% tween-20) and incubated with anti-p65 antibody (ab16502, Abcam), anti-p-p65 antibody (ab86299, Abcam), anti-TLR4 antibody (19811-1-AP, ProteinTech), anti-p-IKBα (ab133462, Abcam), anti-IKBα (ab32518, Abcam), anti-p-IKKα (ab38515, Abcam), anti-IKKα (ab32041, Abcam), or anti-β-actin antibody (60008-1-Ig, ProteinTech) at 4°C overnight. Then, the corresponding secondary antibodies (1 : 5000, ProteinTech) conjugated with HRP were applied for 90 min at room temperature. The membranes were exposed to ECL (#6883, CST Signal Fire™ ECL). The bands were analyzed by the ImageJ software. The levels of target proteins were normalized by β-actin.
Ab133462
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab133462 is a primary antibody targeting the protein of interest. It is suitable for use in various immunoassay applications. The product details and technical specifications are available on the Abcam website.
Automatically generated - may contain errors
Lab products found in correlation
Ab32518, by Abcam (40 mentions)
Ab16502, by Abcam (21 mentions)
Ab32536, by Abcam (20 mentions)
Ab86299, by Abcam (16 mentions)
Ab76302, by Abcam (15 mentions)
Ab8245, by Abcam (9 mentions)
Ab32041, by Abcam (7 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
Ab124957, by Abcam (6 mentions)
Ab205718, by Abcam (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Ab32503, by Abcam (5 mentions)
Ab1791, by Abcam (5 mentions)
Ab181602, by Abcam (5 mentions)
Ab6721, by Abcam (5 mentions)
Ab194528, by Abcam (5 mentions)
Ab38515, by Abcam (4 mentions)
Ab8226, by Abcam (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
71 protocols using ab133462
1
Protein Expression Analysis in Lung Tissues
2
Protein Expression Analysis in NCM460 Cells
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Proteins from NCM460 cells were separated with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China), and the concentration was evaluated through bicinchoninic acid assay (BCA) kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). 22, (link)23 (link) Protein electrophoresis was done through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China). After blocking, the membranes were incubated with the following primary antibodies at 4°C for 12 h: Bax (1:1000; ab32503; Abcam, Shanghai, China), Bcl-2 (1:2000; ab182858), NLRP3 (1:1000; ab263899), p-IκBα (1:10,000; ab133462), IκBα (1:1000; ab32518), p-P65 (1:2000; ab86299), P65 (0.5 µg/mL; ab16502), and β-actin (1:1000; ab8227). Next, the secondary antibodies (1:2000; ab7090) were supplemented into membranes. β-actin acted as an internal reference. Eventually, the blots were assessed using chemiluminescence detection kit (Thermo Fisher Scientific).
3
Molecular Mechanisms of Podocyte Injury
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SAA was provided by Shandong Target Drug Research Co. Ltd (Shandong, China). Prednisone acetate was the product of Zhejiang Xianju Pharmaceutical Co. Ltd. (Zhejiang, China). Doxorubicin hydrochloride for injection was produced by Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). The antibodies used in this study were anti-NF-κB p65 (ab16502, Abcam), anti-inhibitor of NF-κB (IκB) α (ab32135, Abcam), anti-phosphorylation-IκBα (p-IκBα, Ser-36, ab133462, Abcam) and anti-podocin (sc-21009, Santa Cruz Biotechnology, Inc.). Goat anti-rabbit IgG was also the product of Abcam. BCA protein assay kit, anti-β-actin antibody, HRP-labeled goat anti-mouse IgG (H+L) and lipid peroxidation malonaldehyde (MDA) assay kit were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Superoxide dismutase (SOD) detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
4
Western Blot Analysis of TGF-β1 Signaling
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Western blot (WB) analysis was performed according to the methods reported by Rong et al. [38 (link)]. Protein concentration, polyacrylamide gel electrophoresis, film rotation, closing, primary antibody incubation, film washing, secondary antibody incubation and enhanced chemiluminescence (ECL) development were all included in this experiment. The antibodies used were: anti-TGF-β1 (ab215715, Abcam (Cambridge, UK)), anti-Smad2 (ab40855, Abcam), anti-Smad3 (ab40854, Abcam), anti-Smad7 (25840-1-AP, Proteintech (Chicago, IL, USA)), anti-α-SMA (ab7817, Abcam), anti-PI3K (ab191606, Abcam), anti-p-Aktser473 (66444-1-Ig, Proteintech), anti-mTOR (ab134903, Abcam), anti-p-IkBα (ab133462, Abcam), anti-p-P65 (ab76302, Abcam) and anti-p-IKKβ (AF3010, Affinity (Melbourne, Australia)).
5
Protein Expression Analysis via Western Blot
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After the application of cyclic stress and treatment according to the abovementioned methods, whole cell extracts were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The PVDF membrane was then incubated with 10 mM TBS with 1.0% Tween 20 and 10% dehydrated skim milk to block nonspecific protein binding. The membranes were subsequently incubated overnight with rabbit polyclonal antibodies against p-IκBα (1 : 1000 dilution, ab133462), P65 (NF-κB; 1 : 1000 dilution, ab19870), p-P65 (p-NF-κB, 1 : 1000 dilution, ab194726) (Abcam, Cambridge, UK), cytochrome C (1 : 750 dilution, AF2047), and GAPDH (1 : 750 dilution, AF1186) (Beyotime) at 4°C. Subsequently, the membranes were washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (111-035-003; 1 : 1000 dilution, Jackson ImmunoResearch, PA) at 37°C for 1 h. The protein bands were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). Protein expression (normalized to that of GAPDH) was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
6
Western Blot Analysis of Signaling Proteins
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After lysing in RIPA buffer, the collected total protein was separated on 12% SDS-PAGE and shifted onto PVDF membranes. 5% nonfat milk powder was added for blocking membranes. Next, the membrane was probed all night at 4 °C with primary antibodies against loading control GAPDH (ab8245, 1/1000; Abcam, Cambridge, MA, USA), TRAP (ab65854, 1/1000; Abcam), NFATc1 (ab175134, 1/1000; Abcam), TRAF3 (ab36988, 1/1000; Abcam), TRAF1 (ab129279, 1/1000; Abcam), TRADD (ab110644, 1/1000; Abcam), TRAF2 (ab230795, 1/1000; Abcam), ADAM17 (ab39162, 1/1000; Abcam), TNFRII (ab8161, 1/1000; Abcam), TNFRSF1A (ab19139, 1/1000; Abcam), p-IKB (ab133462, 1/1000; Abcam), IKB (ab32518, 1/1000; Abcam), TNFRSF11 (NB100-56508, 1/1000; Novus Biologicals, Littleton, CO, USA), p-IKKα (ab38515, 1/1000; Abcam), IKKα (ab32041, 1/1000; Abcam), p-IKKβ (ab59195, 1/1000; Abcam), IKKβ (ab124957, 1/1000; Abcam), p-Erk (ab214036, 1/1000; Abcam), Erk (ab184699, 1/10000; Abcam), p-MEK (ab96379, 1/1000; Abcam), MEK (ab32091, 1/1000; Abcam). On the following day, the HRP-secondary antibodies were added for 2 h at room temperature. The protein density was examined by the ECL luminous liquid (Pierce, Rockford, IL, USA). The assay was taken in triplicate.
7
Brain Tissue Protein Analysis
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Total proteins in the brain tissues were extracted in RIPA lysis buffer (Beyotime), and the protein concentration was measured by a BCA protein concentration kit (Beyotime). The proteins were then separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electro‐transferred onto the polyvinylidene difluoride membrane (Millipore). After being blocked with 5% skimmed milk for 1 h at room temperature, the membranes were firstly incubated with primary antibodies, including anti‐p‐IKKβ (1:500; ab194519; Abcam), anti‐IKKβ (1:1000; ab124957; Abcam), anti‐p‐IkBα (1:3000; ab133462; Abcam), anti‐IkBα (1:1000; ab32518; Abcam), anti‐NF‐κB p65 (1:1000; ab16502; Abcam), anti‐NF‐κB p‐p65 (S536) (1:500; ab86299; Abcam), anti‐Nrf2 (1:1000; ab62352; Abcam), anti‐HO‐1 (1:500; ab13248; Abcam), anti‐NQO1 (1:500; ab80588; Abcam), anti‐Histone 3 (1:1000; ab1791; Abcam), and anti‐β‐actin (1:1000; ab8226; Abcam), at 4℃ overnight and then with corresponding secondary antibodies (1:3000; Beyotime) at room temperature for 1 h. The blotted protein bands were visualized by an enhanced chemiluminescence kit (Beyotime, Shanghai, China). Image J software (NIH Image) was used to analyze the gray level of the bands.
8
Western Blot Analysis of Testis Proteins
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Testis tissues were lysed in ice-cold RIPA buffer (Beyotime Institute of Biotechnology, Hangzhou, China) for 30 min, the lysates were centrifuged at 12,000 × g for 10 min, and then the supernatant solution was collected. The protein concentration of the supernatant was detected with a BCA kit (Solarbio). Protein samples were then loaded on 5% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 2 h at room temperature and then washed three times with TBST. Next, the membranes were incubated with the specific primary anti-bodies (Abcam, United States) including anti-TP53 (ab202026, 1:2000), p-AKT1 (ab81283, 1:5000), TNF-α (ab6671, 1:1000), p-NF-κB p65 (ab222494, 1:1000), p-IκBA (ab133462, 1:5000), Bcl-XL (ab32370, 1:1000), Bcl-2 (ab32124, 1:1000), Bax (ab32503, 1:5000), cleaved-Caspase 3 (ab32042, 1:1000), and cleaved-Caspase 9 (ab2324, 1:2000) at 4 C overnight. Afterwards, the membranes were washed with TBST and further incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit lgG antibody (ab205718, 1:2000) for 1 h at room temperature. The protein bands were visualized with an enhanced chemiluminescence (ECL) system, and the relative intensity of the band was quantified with Image J software (National Institutes of Health, United States). β-actin was used as the internal control to normalize the relative band density.
9
Western Blot Analysis of Protein Expression
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Total protein of the cells was extracted with total protein extraction (Millipore, Bedford, MA, USA). Protein concentration was determined using the BCA protein assay kit. Cell lysates containing an equivalent amount of protein (10 μg) were electrophoresed through 12% SDS polyacrylamide gels, and subsequently transferred onto a nitrocellulose membrane (Sigma-Aldrich, Shanghai, China). Western blot analysis was performed with P-Glycoprotein Monoclonal Antibody (F4), (1 : 100; Thermo Fisher scientific), anti-IKB alpha (ab32518) (1 : 5000; Abcam), anti-IKB alpha (phospho S36) (ab133462), (1 : 50 000; Abcam), NFkB p50 Monoclonal Antibody (5D10D11) (1 : 1000; Thermo Fisher scientific), anti-NF-κB p65 (ab16502) (1 : 5000; Abcam), GAPDH was used as an internal control. Afterwards, membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were photoed with ChemiDoc system (Bio-Rad). The expression levels of these proteins were quantified with Image J software.
10
Western Blot Analysis of TLR4 Signaling
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Western blot analysis was performed as previously described (32 (link),33 (link)). Total protein was extracted using RIPA buffer (Beyotime Institute of Biotechnology). The extraction and isolation of nuclear and cytoplasmic proteins were performed using the Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology). The concentrations of total cellular protein were determined using a BCA assay kit (Pierce; Thermo Fisher Scientific, Inc.). Briefly, 40 µg extracted protein samples were transferred onto polyvinylidene difluoride (PVDF, Millipore) membranes and then blocked with 5% skim milk at 4°C overnight. Each membrane was then probed with primary antibodies against TLR4 (1:2,000, ab9870), nuclear p-p65 (1:1,500, ab53489), p65 (1:2,000, ab86299), p-IκBα (1:2,000, ab133462), IκBα (1:2,000, ab32518), MyD88 (1:1,000, ab199247), Histone 3 (1:1,000, ab1791) and β-actin (1:1,000, ab8226) (all from Abcam) at 4°C overnight, followed by HRP-conjugated goat anti-rabbit IgG (1:10,000; cat. no. ab205718; Abcam). The protein bands were developed using an ECL kit (GE Healthcare) and blot bands were quantified using ImageJ software version 1.46 (Rawak Software, Inc.).
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