Total cell lysate was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology). Approximately 30 µg of protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk, the membranes were incubated overnight with primary antibodies against CEP55 (Abcam, ab170414), MMP2 (Abcam, ab37150), MMP9 (Abcam, ab38898), Cyclin D1 (Abcam, ab134175), p-IKKβ (Abcam, ab59195), IKKβ (Abcam, ab32135) and IκBα (Abcam, ab32518), followed by incubation with secondary antibodies. Human α-tubulin (Abcam, ab7291) or GAPDH (Abcam, ab8245) were used as the endogenous references, according to the details. Immunoreactive protein bands were visualised using the ECL method (Invitrogen), according to the manufacturer’s recommendations.
Ab32135
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab32135 is a primary antibody for use in immunoassays. It is a rabbit polyclonal antibody that recognizes a specific target protein. The product is supplied in a liquid format.
Automatically generated - may contain errors
Lab products found in correlation
Ab32518, by Abcam (4 mentions)
Ab16502, by Abcam (3 mentions)
Ab133462, by Abcam (3 mentions)
Ab194528, by Abcam (3 mentions)
Ab76302, by Abcam (3 mentions)
Ab8245, by Abcam (2 mentions)
Ab13847, by Abcam (2 mentions)
Ab32536, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab7291, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Ecl method, by Thermo Fisher Scientific (1 mentions)
Ab37150, by Abcam (1 mentions)
Ab170414, by Abcam (1 mentions)
Ab59195, by Abcam (1 mentions)
Ab38898, by Abcam (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
Superoxide dismutase sod detection kit, by Nanjing Jiancheng (1 mentions)
Anti β actin antibody, by Beyotime (1 mentions)
Hrp labeled goat anti mouse igg h l, by Beyotime (1 mentions)
9 protocols using ab32135
1
Western Blot Analysis of Protein Expression
2
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).
3
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Sigma, Shanghai, China) was applied to extract total proteins. A BCA kit (Sigma) was employed to measure the concentration of protein in the supernatant. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis was used to separate proteins, followed by their electrical transfer to a polyvinylidene fluoride membrane. Post blocking with 5% BSA, membranes were kept overnight at 4 °C alongside diluted primary antibodies: anti-USP11 (1:5000, ab109232, Abcam), anti-NLRP3 (1:1000, ab263899, Abcam, Cambridge, UK), anti-GSDMD-N (1:1000, ab215203, Abcam), anti-caspase-1 (1:1000, ab207802, Abcam), anti-IL-1β (1:1000, ab300501, Abcam), anti-IL-18 (1:1000, ab207323, Abcam), anti-p-IKKβ (1:1000, ab194528, Abcam), anti-IKKβ (1:500, ab32135, Abcam), anti-p-NF-kB (1:1000, ab76302, Abcam), anti-NF-kB (1:1000, ab16502, Abcam), anti-TRAF3 (1:1000, ab155298, Abcam), and anti-β-actin (1:1000, ab8227, Abcam) and then incubation for 1 h by secondary antibody. An ECL chromogenic substrate was used to visualize the bands.
4
Western Blot Analysis of Apoptotic Markers
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The neurons were lysed, pulverized, and centrifuged at 15000 r/min for 15 min. Protein samples in the supernatant of neurons were separated by sodium lauryl sulfate-polyacrylamide gels (Beyotime, Shanghai, China) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blockage of the PVDF membranes for 1 h, protein samples were incubated with rabbit-anti-mouse primary antibodies against Bcl-2 (1:2000, Catalog No.: ab182858, Abcam, Cambridge, MA, USA), Bax (1:1000, Catalog No.: ab32503; Abcam), Caspase-3 (1:500, Catalog No.: ab13847; Abcam), Caspase-9 (1:2000, Catalog No.: ab202068; Abcam), p53 (1:1000, Catalog No.: ab131442; Abcam), Fas (1:1000, Catalog No.: ab82419; Abcam), NF-κB p65 (1:2000, Catalog No.: ab32536; Abcam), IκBα (1:4000, Catalog No.: ab32518; Abcam), pIκBα (1:1000, Catalog No.: 2859, Cell Signaling Technology, Danvers, MA, USA), IKKβ (1:5000, Catalog No.: ab32135; Abcam) and GAPDH (1:2000, Catalog No.: ab8245; Abcam) at 4℃ overnight and then with goat anti-rabbit IgG II antibodies (1: 5000, Catalog No.: ab6721; Abcam).
5
NF-κB Activation Pathway Evaluation
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Antibodies against Iκκβ (ab32135) and p65 (ab7970) and Alexa Fluor 555 Donkey anti-Rb IgG (H + L) antibody (ab150074) were obtained from Abcam (Cambridge, MA, USA). The other antibodies used were as follows: anti-IKB-α (sc-1643) and anti-p-IKB-α (sc-8404) (Santa Cruz Biotechnology, CA, USA), anti-p-p65 (3033S; Cell Signaling Technology, Danvers, MA, USA), and anti-NFKB1 (NF-κB p50) (14220-1-AP; ProteinTech, Rosemont, IL, USA). Anti-GAPDH (H301; TransGen Biotech, Beijing, China) was used as normalized control. Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (CSB-E04638h), IL-8 (CSB-e04641h), and TNF-α (CSB-EQ023955HU) were obtained from Cusabio Biotech (Wuhan, China). M-MLV Reverse Transcriptase (M1701) and GoTaq qPCR Master Mix (A6001) were obtained from Promega (Madison, WI, USA).
6
Western Blot Analysis of NF-κB Signaling
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At end of incubations/exposure to agents, cells were lysed in pre-heated Laemmli’s sample buffer, dispered by repeating passing through a 21G needle and heated at 100°C for 5 min prior to analysis. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to separate proteins, which were then transferred to nitrocellulose membranes by electrophoretic blotting in wet conditions. Non-specific binding then blocked in 3% (w/v) BSA in NaTT buffer and membranes were incubated overnight, either at room temperature or 4°C, in primary antibody specific to the target protein diluted to optimal concentration in NaTT buffer containing 0.3% (w/v) BSA. Antibodies and dilutions used were as follows: p65 (1:10000, sc-8008, Santa Cruz), p-p65 (1:1000, #3031, Cell Signaling), IκBα (1:7500, #1242, Cell Signaling), IKKβ (1:2500, ab32135, Abcam) and β-tubulin (at 1:5000, ab21058, Abcam).
Membranes were incubated in secondary HRP-conjugated antibody (either rabbit or mouse, depending on primary antibody) diluted 1:10000. Enhanced chemiluminescence (ECL) reagent was used to detect presence of the antibody and X-ray films developed using an X-OMAT machine (Kodak) or the G:Box imaging system (SynGene).
Membranes were incubated in secondary HRP-conjugated antibody (either rabbit or mouse, depending on primary antibody) diluted 1:10000. Enhanced chemiluminescence (ECL) reagent was used to detect presence of the antibody and X-ray films developed using an X-OMAT machine (Kodak) or the G:Box imaging system (SynGene).
7
Western Blot Analysis of NF-κB Pathway
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Cells lysed with radioimmunoprecipitation assay (RIPA) lysis buffer were centrifuged at 12000 g for 20 min at 4 °C. Protein concentrations in the collected supernatant were quantified with a BCA assay kit. After equal amounts of protein (20 μg/well) were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they were transferred onto polyvinylidene difluoride (PVDF) membranes and incubated in 5% BSA for 1 h at room temperature. The membranes were washed with Tris-borate saline containing 0.1% Tween-20 (TBST) and were incubated with primary antibodies against FGL1 (1:1000 dilution, 16000-1-AP, Proteintech, United States), IKKα (1:1000 dilution, ab32041, Abcam, United Kingdom), IKKβ (1:1000 dilution, ab32135, Abcam), p-IKKα/β (1:1000 dilution, ab194528, Abcam), IκBα (1:1000 dilution, ab32518, Abcam), p-IκBα (1:1000 dilution, ab133462, Abcam), NF-κB (p65, 1:1000 dilution, ab32536, Abcam), p-p65 (1:1000 dilution, ab76302, Abcam) and β-actin (1:1000 dilution, 20536-1-AP, Proteintech) at 4 °C overnight. The membranes were washed with TBST again and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The blots were imaged using enhanced chemiluminescence (ECL). ImageJ software was used to calculate the protein signal grey values.
8
Anti-inflammatory Effects of DHA Lipid Mediators
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FO (EPA C20:5 n-3: 60 mg/g, DHA, C22:6, n-3: 460 mg/g, DPA, C22:5 n-3 + n-6, 105 mg/g) was acquired from KD Nutra (Branttvag, Norway). A mixture of lipid mediators (LM) was obtained from DHA through enzymatic reaction using soybean lipoxygenase, which is composed of 17S-monohydroxy DHA, resolvin D5, and protectin DX at ratio of 3:47:50. Palmitic acid (PA) was purchased from Fluka (Seelze, Germany). Human liver cancer cell line HepG2 was purchased from Korea Cell Line Bank (Seoul, Republic of Korea). A bicinchoninic acid (BCA) assay kit was purchased from Sango Company (San Diego, CA, USA). Antibodies against IKKB (ab32135), p-IKKB (ab194528), IKB (ab32518), p-IKB (ab133462), NF-kB (p65) (ab16502), p-NF-kB (p-p65) (ab76302), and GAPDH (ab181602) were purchased from Abcam (Cambridge, MA, USA). Enhanced chemiluminescence (ECL) was purchased from Bio-Rad Laboratories, Inc. (Tewksbury, MA, USA). The assay kits for triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol were purchased from BioVision (Milpitas, CA, USA). Mouse/human IL-6 and TNF-α ELISA kits were purchased from Abcam (Cambridge, MA, USA).
9
Antibody-Based Immunoblot and Immunofluorescence Analysis
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The following antibodies were used for immunoblot analysis: rabbit anti-IKKβ [Y466] (ab32135, 1:2000; Abcam, Cambridge, United Kingdom), goat anti-Lcn2 (AF1857, 1:1000; R&D Systems, Minneapolis, MN, USA), rabbit anti-glial fibrillary acidic protein (GFAP; ab7779, 1:1000; Abcam), rabbit anti-Bax (2772, 1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Bad (9292, 1:1000; Cell Signaling Technology), rabbit anti-NF-κB p65 (C20, sc-372, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-phospho NF-κB p65 (Ser536, 3033, 1:1000; Cell Signaling Technology), rabbit anti-GAPDH (FL-335, (sc-25778, 1:2000; Santa Cruz Biotechnology), rabbit anti-β-tubulin (ab6046 1:10000; Abcam) and HRP-conjugated goat anti-rabbit or donkey anti-goat were obtained from Santa Cruz Biotechnology.
For immunofluorescence, the following primary antibodies were used: mouse anti-NeuN (MAB377, 1:300; Millipore-Sigma), rabbit anti-cleaved caspase 3 (ab13847, 1:400; Abcam), and mouse anti-tubulin-β3 (TUJ1) (MMS-435P, 1:500; BioLegend, San Diego, CA, USA). Corresponding Alexa Fluor-conjugated secondary antibodies were obtained from Thermo Fisher Scientific, and DAPI was purchased from Merck (Darmstadt, Germany).
For immunofluorescence, the following primary antibodies were used: mouse anti-NeuN (MAB377, 1:300; Millipore-Sigma), rabbit anti-cleaved caspase 3 (ab13847, 1:400; Abcam), and mouse anti-tubulin-β3 (TUJ1) (MMS-435P, 1:500; BioLegend, San Diego, CA, USA). Corresponding Alexa Fluor-conjugated secondary antibodies were obtained from Thermo Fisher Scientific, and DAPI was purchased from Merck (Darmstadt, Germany).
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