Western blots were performed as previously described [25 (link)]. Total protein was extracted from kidney tissue in RIPA lysis buffer (25 mM Tris-HCl, 25 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, and 0.1% SDS) with 1% PMSF protease inhibitors (P1005, Beyotime Biotechnology, China) and phosphatase inhibitors (P1081, Beyotime Biotechnology, China) added. Equal amounts of protein were separated by 10-15% SDS-PAGE and then transferred to PVDF membranes (IPVH00010, Millipore, Germany). After blocking with 5% nonfat milk for 3 h, the membranes were incubated with the primary antibodies including TGF-β1, α-SMA, Nrf2, HO-1, NQO1, ASC, caspase-1 (ab215715, ab32575, ab137550, ab13248, ab80588, ab175449, ab1872, Abcam, UK), p-Smad2, Smad2, p-Smad3, Smad3, E-cadherin, NLRP3 ((#18338, #5339, #9520, #9523, #3195, #15101, Cell Signaling Technology, USA), and cleaved caspase-1 (AF4005, Affinity Biosciences, USA) at 4°C overnight. Then, the membranes were washed 3 times with TBST and incubated with the secondary antibodies for 1 h at room temperature. After washing with TBST for a further three times, the protein bands were visualized by enhanced chemiluminescence (32134, Thermo, USA) solution and imaged with Automated Imaging System (Gene Gnome5, Synoptics Ltd, UK). GAPDH or β-actin was assumed to be similarly abundant in all samples and was used as a loading control.
Ab175449
Manufactured by Abcam
Sourced in United States
Ab175449 is a lab equipment product offered by Abcam. It is a device used for scientific research purposes. No further details on its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab263899, by Abcam (4 mentions)
Ab214185, by Abcam (4 mentions)
Ab1872, by Abcam (3 mentions)
Ab138483, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab234437, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab109234, by Abcam (2 mentions)
Ab58705, by Abcam (2 mentions)
Ab219800, by Abcam (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Ab80588, by Abcam (1 mentions)
Ab13248, by Abcam (1 mentions)
Ab215715, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Af4005, by Affinity Biosciences (1 mentions)
P1081, by Beyotime (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
13 protocols using ab175449
1
Western Blot Analysis of Kidney Proteins
2
Immunoprecipitation and Western Blot Analysis of Caspase-8 and ASC
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Total protein was extracted from each group as stated above and stored at −80 °C. Incubated with 1 μl anti-caspase-8 or ASC antibody overnight at 4 °C was 50 μl of protein. This reaction mixture was then incubated with protein A magnetic beads (2366538, Millipore, Temecula, CA, USA) for 30 min at 4 °C. Precipitates were washed three times with washing buffer and then eluted from protein A magnetic beads by boiling with 1 × SDS for 10 min at 90–100 °C. Western blot analysis was used to evaluate the expression of caspase-8 and ASC. Immunoprecipitation antibody, anti-caspase-8 (#8592, Cell Signaling Technology, Beverly, MA), anti-ASC (sc-22513,Santa Cruz, CA, USA); Western blot analysis anti-caspase-8 (sc-6134, Santa Cruz, CA, USA), anti-ASC (ab175449, Abcam), homophytic IgG as the negative control.
3
Immunoblot Analysis of Inflammatory Mediators
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We extracted and denatured the total protein in each group of cells and tissues. After SDS-gelelectrophoresis, we transferred them to nitrocellulose membranes. Then, we incubated the different primary antibodies including TLR4 (1:500, ab13556, Abcam, UK), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam), IL-18 (1:4000, 60,070-1-Ig, Proteintech, USA), caspase-1 (1:1000, 22,915-1-AP, Proteintech), NF-κB (1:2000, 10,745-1-AP, Proteintech), ASC (1ug/mL, ab175449, Abcam), Pro-MMP9 (2ug/mL, MAB9111, R&D), Claudin5 (1:4000, ab131259, Abcam) and β-actin(1:5000, 66,009-1-Ig, Proteintech)overnight 4 °C.After washing the membranes three times, the blots were incubated with fluorescent-basedanti-rabbit(1:6000, SA00001-2, Proteintech), anti-mouse(1:5000, SA00001-1, Proteintech) or anti-goat(1:5000, SA00001-4, Proteintech) IgG secondary antibody at room temperature.We visualized the proteins by chemiluminescence detection reagent (Thermo Fisher, Waltham, MA, USA). We used Image J software to analyze the gray value of all bands quantitatively.
4
Hippocampal Protein Expression Analysis
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One day after the behavioral tests, the rats were anesthetized with 10% chloral hydrate (4 mL/kg) and the tissues were rapidly collected. For western blotting analysis, total protein was prepared from the right hippocampus, and the protein concentrations were analyzed using Bradford method. The samples were loaded on precast 12% SDS-PAGE gels with approximately 50 μg protein in each lane. The following antibodies and concentrations were used over night at 4 °C; LC3 (Cell signaling, 4108; 1:1000), Beclin-1 (Abcam, ab62557; 1:1000), NLRP3 (Abcam, ab214185; 1:200), ASC (Abcam, ab175449; 1:1000), caspase-1 (Abcam, ab1872; 1:1000), IL-1β (Abcam, ab9722; 1:1000), and β-actin (Proteintech, 66009-1-Ig; 1:4000). The signals were normalized to β-actin as an internal standard. It was then probed with HRP-conjugated secondary antibody for 40 min. The film signals were digitally scanned and then quantified using ImageJ software. The signals were normalized to β-actin as an internal standard.
5
Western Blot Analysis of NLRP3 Inflammasome Pathway
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Primary antibodies used in this study were anti‐NLRP3 (ab263899, 1:800; Abcam), anti‐ASC (ab175449, 1:500; Abcam), anti‐caspase‐1 (ab138483, 1 µg/mL; Abcam), anti‐N‐GSDMD (ab215203, 1:800; Abcam), anti‐inhibitor a of NF‐κB (IκBα) (#4814S, 1:800; Cell Signaling Technology), anti‐p‐IκBα (#2859, 1:800; Cell Signaling Technology), anti‐p65 (ab32536, 1:800; Abcam), LaminB1 (ab16048, 0.1 µg/mL; Abcam), and anti‐GAPDH (ab8245, 1:1000; Abcam). The nuclear and cytoplasmic proteins from RAW264.7 cells were isolated with a nuclear extraction kit (P0027; Beyotime). The total protein concentration in each cell lysate was determined with a BCA Protein Assay Kit (Beyotime). Subsequently, the proteins were separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 60 min and incubated with primary antibodies at 4°C overnight. Next day, the membranes were incubated with secondary antibodies (ab6721, 1: 2000; Abcam) at room temperature for 1.5 h. Finally, the protein expression was determined by an ECL kit and Scion Image v. 4.0.2 software (Scion Corporation).
6
Visualizing Colon Immune Responses
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Immunofluorescence staining of mouse colon sections was performed as previously described (26 (link)). The sections were incubated with primary antibodies against VDR (ab109234, Abcam), NLRP6 (ab58705, Abcam), Caspase-1 (ab138483, Abcam) and ASC (ab175449, Abcam) at 4°C overnight, followed by an incubation with fluorescently labelled secondary antibodies. The sections were examined using a Leica confocal microscope (LEICA TCS SP5).
7
Quantification of NLRP3 Inflammasome Proteins
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The expression of NLRP3, ASC, caspase‐1, IL‐1β, and TGF‐β1 was measured by Western blot analysis. Total proteins in ventricular chamber tissues were isolated using radioimmunoprecipitation assay buffer, and concentrations were measured using BCA Protein Assay Kits (Beyotime Institute Biotechnology). Total protein was separated by 10% polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Nonspecific binding was blocked with 5% skim milk in Tris‐buffered saline Tween‐20, then the membranes were incubated for 4 h at 37°C with primary antibodies against NLRP3 (ab263899, 1:1000), ASC (ab175449, 1 μg/ml), caspase‐1 (ab207802, 1:1000), IL‐1β (ab9722, 0.1 μg/ml), TGF‐β1 (ab179695, 1:1000), and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (ab181602, 1:10000) (Abcam), followed by the corresponding secondary antibodies (ab205718/ab205723, Abcam) for 1 h. The membranes were rinsed, then proteins were visualized using enhanced chemiluminescence. Relative protein expression was calculated as the ratio of target protein expression to that of the reference protein (GAPDH). The experiment was repeated three times and the data were averaged.
8
Western Blot Analysis of NLRP3 Inflammasome
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Lysates from tissues and cells were prepared using RIPA lysis buffer and were quantified using a BCA kit (cat. #BCA1; Sigma–Aldrich). Afterwards, lysates were resolved in SDS-PAGE to separate proteins and were then transferred to polyvinylidene fluoride membranes. The membranes were immersed in 5% skimmed milk to block for non-specific binding and labelled with anti-WHSC1 (1:2000; cat. #ab223694; Abcam), NLRP3 (1:500; cat. #ab214185; Abcam), ASC (1:2000; cat. #ab175449; Abcam), caspase-1 p20 (1:1000; cat. #PA5-99390; Thermo Fisher Scientific), IL-18 (1:1000; cat. #ab191860; Abcam), IL-1β (1:1000; cat. #ab234437; Abcam), GSDMD FL (full length GSDMD; 1:1000; cat. #ab219800; Abcam), GSDMD CL (GSDMD-N terminal segment; 1:1000; cat. #10137S; Cell Signaling Technology, Danvers, MA), NEK7 (1:3000; cat. #ab133514; Abcam) and β-actin (1:5000; cat. #ab8227; Abcam) Abs at 4°C for 12 h. Afterwards, the membranes were incubated with IgG H&L (HRP; 1:5000; cat. #ab6728; Abcam) for 2 h at room temperature. β-Actin was designated as the internal control protein. Densitometry of bands was measured utilising the ECL Western Blotting Substrate (cat. #32132X3; Thermo Fisher Scientific).
9
Quantitative Western Blot Analysis of Myocardial Proteins
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The total protein content from myocardial tissue or cells was extracted for measurement of protein concentration using the bicinchoninic acid assay (ThermoFisher). The extracted protein was added into the loading buffer and boiled at 95 °C for 10 min. Next, the protein sample (30 μg) was separated by 10% (w/v) electrophpresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked using 5% BSA for 1 h and cultured with the primary antibodies at 4 °C overnight. After being washed by tris buffered saline tween (TBST), the membranes were incubated with secondary antibody for 1 h and then washed by TBST before enhanced chemiluminescence developing and visualization using Bio-Rad GelEZ imager (Bio-Rad, Inc., Hercules, CA, USA). Image J software (National Institutes of Health, Bethesda, Maryland, USA) was utilized to analyze the gray value of target bands. The antibodies used were IP3R1 (1:2000, ab264281, Abcam), IgG (1:2000, ab205718, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab175449, Abcam), GSDMD (1:2000, ab219800, Abcam), GAPDH (1:1000, ab8245, Abcam), and ERP44 (1:1000, #3798, Cell Signaling Technology, Danvers, MA, USA).
10
Quantification and Immunoblotting of Colon Proteins
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The collected MIECs and mouse colon tissues were extracted using protein lysis buffer (Sigma-Aldrich, USA) and quantified via a bicinchoninic acid assay (Pierce, USA). Protein samples were then electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF, EMD Millipore, MA, USA), which was probed with antibodies against VDR (ab109234, Abcam), NLRP6 (ab58705, Abcam), Caspase-1 (ab138483, Abcam) and ASC (ab175449, Abcam) at a dilution of 1:1000. Blots were subsequently detected and visualized using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA) according to protocols provided by the manufacturer. A Bio-Rad scanning system was used to detect immunoreactive protein bands, and GAPDH (ab204276, Abcam) was used as a control.
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