After the treatments, the cells were lysed on ice for 20 min with a mixture of 100 μl RIPA buffer (Fdbio science, Hangzhou, China), 1 μl protease inhibitor (Fdbio science) and 1 μl phosphatase inhibitor (Fdbio science) and centrifuged at 14,000 g for 25 min. Protein concentrations were determined using a BCA Total Protein Assay Kit (CWBIO, China). Protein samples (40 μg/lane) were separated on SDS-PAGE gels (8% or 12%, Epizyme, China) and electrotransferred to PVDF membranes. The blots were incubated with the following primary antibodies: anti-NLRP3 (1:500, A12694, ABclonal), anti-GSDMD (1:500, 39754, CST), anti-p20 (1:500, AF4005, Affinity), anti-CHOP (1:500, ab11419, Abcam), anti-GRP78 (1:500, ab21685, Abcam), anti-COL2A (1:500, ab34712, Abcam), anti-Aggrecan (1:500, ab36861, Abcam), anti-MMP3 (1:500, ab52915, Abcam), anti-MMP13 (1:500, ab39012, Abcam), anti-ADAMTS5 (1:1000, ab41037, Abcam), and anti-SREBP1 (1:1000, ab28481, Abcam). The membranes were washed and incubated with the HRP-conjugated secondary antibodies (1:8000, ABclonal, China) for 1 h. The bands were developed with chemiluminescence reagents and imaged by a myECL imager (Syngene G:BOX ChemiXT4, United Kingdom). The experiment was repeated in triplicate, and the blots were quantified by ImageJ software. An antibody against β-actin (AC026, 1:1000; ABclonal) served as an endogenous control.
A12694
Manufactured by ABclonal
Sourced in United States
A12694 is a lab equipment product from ABclonal. It is designed for laboratory use, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
A0964, by ABclonal (3 mentions)
Ac026, by ABclonal (2 mentions)
Ab34712, by Abcam (2 mentions)
Protease inhibitor, by Fdbio (1 mentions)
Phosphatase inhibitor, by Fdbio (1 mentions)
Ripa buffer, by Fdbio (1 mentions)
Af4005, by Affinity Biosciences (1 mentions)
Ab21685, by Abcam (1 mentions)
G box chemi xt4, by Syngene (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab36861, by Abcam (1 mentions)
Ab11419, by Abcam (1 mentions)
Ab28481, by Abcam (1 mentions)
Ab52915, by Abcam (1 mentions)
Ab41037, by Abcam (1 mentions)
Nbp2 15286, by Novus Biologicals (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
A16672, by ABclonal (1 mentions)
Wl03325, by Wanlei (1 mentions)
Wl01980, by Wanlei (1 mentions)
6 protocols using a12694
1
Comprehensive Protein Analysis of Cellular Responses
2
Histological Analysis of Knee Cartilage
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After 6-week intra-articular injection, the complete right knee of every group (n = 3/group) was collected for the histologic section, and the procedures were in accordance with the histological examination. After deparaffinization, the sections were rinsed with 0.3% H2O2 in 60% methanol for 30 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 20 min. The section then was incubated in goat serum in PBS for nonspecific adsorption. For immunohistochemistry, sections were incubated with (1) anti-rabbit polyclonal antibody directed at collagen II (1 : 400; Abcam, ab34712), (2) anti-MMP-13 mouse monoclonal antibody (1 : 150; Novus, OTI2D8), (3) anti-ADAMTS-5 rabbit polyclonal antibody (1 : 100; Novus, NBP2-15286), (4) anti-NLRP3 rabbit polyclonal antibody (1 : 200; ABclonal, A12694), or anti-caspase-1 rabbit polyclonal antibody (1 : 150; ABclonal, A0964). Horseradish peroxidase- (HRP-) conjugated secondary antibody was applied and stained with a diaminobenzidine (DAB) kit. The positive stained chondrocytes in three central regions of articular cartilage were counted using Image-Pro Plus version 6.0 software.
3
Protein Expression Analysis in Ischemic Brain
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The protein extracts of brain tissue were prepared as described previously. To determine NLRP3, ASC, and cleaved caspase-1 protein expressions in the ischemic core of the brain (Fig. 2 A), the protein concentrations were measured using BCA assay. Protein samples were separated by SDS-PAGE and were transferred onto polyvinylidene fluoride membranes. The membrane was blocked with 5% nonfat milk in TBST (0.1% Tween 20 in TBS) for 2 hours at room temperature and incubated with primary antibodies of anti-NLRP3 (1:1000, A12694; ABclonal Technology, Wuhan, China), anti-ASC (1:1000, A16672; ABclonal Technology, Wuhan, China), anti-cleaved caspase-1 (1:1000, WL03325; Wanleibio, Shenyang, China), and anti-NF-κB (1:500, WL01980; Wanleibio, Shenyang, China) at 4°C overnight. After being washed 3 times, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) secondary antibodies (1:5000, AB0101; Abways Technology, Shanghai, China) for 2 hours. Then, the blots were visualized by enhanced chemiluminescence detection system (Chemidoc XRS+, Bio-Rad; Berkeley, CA). The results were normalized to anti-β-actin (1:1000, AY0573; Abways Technology, Shanghai, China) or anti-H3 (1:500, WL09804a; Wanleibio, Shenyang, China) and the intensity of bands was quantified by ImageJ.
4
Hippocampus and Intestinal Protein Analysis
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Western blot analysis was performed on the dorsal hippocampus and intestinal tissue samples. Briefly, proteins of the dorsal hippocampus and intestines were isolated, separated by electrophoresis, transferred to a PVDF membrane, and probed with the following primary antibodies: NLRP3 (1:1000, A12694, ABclonal, Woburn, MA, USA), caspase 1 (1:1000, ZRB1233, Sigma-Aldrich, Burlington, MA, USA), caspase 3 (1:1000, sc-56053, Santa Cruz Biotechnology, Dallas, TX, USA), TLR4 (1:1000, SC-293072, Santa Cruz Biotechnology, Dallas, TX, USA), claudin-1 (1:1000, ab15098, Cambridge, MA, USA), occludin (1:1000, SC-8144, Santa Cruz Biotechnology, Dallas, TX, USA), and ZO-1 (1:1000, ab96587, Cambridge, MA, USA). The integrated optical density (IOD) was factored into Ponceau S staining to correct for any variations in total protein loading, as we previously reported [13 (link)]. Bands of interest were visualized using electrochemiluminescence reagents (PerkinElmer, Waltham, MA, USA) and quantified by densitometry (Quantity One Analysis software; Bio-Rad, Hercules, CA, USA) as the IOD after subtraction of the background. The protein abundance was represented as IOD/Ponceau S.
5
Western Blot Analysis of Inflammasome Proteins
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To detect activated inflammasome proteins, INS-1 cells were stimulated with 1 µL LPS/200 μM PA–BSA for 4 h [24 (link),59 (link)]. Total protein extraction was performed using ice-cold NP-40 (1.0% NP-40, 150 mM NaCl, 50 mM Tris-Cl, pH 8.0) lysis buffer containing a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). A Western blot analysis was performed as previously described [58 (link)] with the following antibodies: MAPK8IP1 (anti-rabbit; 1:1000, #Ab24449, Abcam, Cambridge, UK), NLRP3 (anti-rabbit; 1:1000, #A12694, Abclonal, Woburn, MA, USA), CASPASE-1 (anti-rabbit; 1:1000, #A0964, Abclonal, Woburn, MA, USA), IL-1β (anti-rabbit; 1:1000, #A162888, Abclonal, USA), GSDMD (anti-rabbit; 1:1000, #A10164, Abclonal, USA), JNK (anti-rabbit; 1:1000, #A48567, Abclonal, USA), pJNK (anti-rabbit; 1:1000, #AP0631, Abclonal, USA), β-actin (anti-mouse, 1:1000, #A5441, Sigma-Aldrich, Darmstadt, Germany), and secondary anti-mouse (#7076S) or anti-rabbit (#7074S, from Cell Signaling Technology, Danvers, MA, USA). Chemiluminescence was detected using the Clarity ECL substrate kit (Bio-Rad, Hercules, CA, USA). β-actin was used as an endogenous control.
6
Immunoblot Assay of Cell Signaling
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Immunoblot assays were performed according to standard protocol. Target proteins were detected with antibodies against NLRP3 (A12694, Abclonal, Wuhan, China), collagen I (A5786, Abclonal, Wuhan, China), fibronectin (A16678, Abclonal, Wuhan, China), Flag (F1804, Sigma, St Louis, USA), caspase1 (A0964, Abclonal, Wuhan, China), IL-1β (A11369, Abclonal, Wuhan, China), IL-18 (A1115, Abclonal, Wuhan, China), phos-smad3 (AP0727, Abclonal, Wuhan, China), E-cadherin (3195, CST, Danvers, USA), N-cadherin (14215, CST, Danvers, USA), FIP1 (A7138, Abclonal, Wuhan, China), β-actin (AC026, Abclonal, Wuhan, China).
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