Total protein was isolated from MPC5 cells using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and quantitated using a BCA protein quantification kit (Abcam, Cambridge, UK), and the proteins were separated via 10% SDS‒PAGE. Then, the proteins were transferred to PVDF membranes (Roche, Basel, Switzerland). After being sealed with 5% nonfat milk and washed three times with TBST, the membranes were incubated with primary antibodies against Bax (1:1,500, ab182733; Abcam, Cambridge, UK), Bcl-2 (1:2,000, ab182858; Abcam, Cambridge, UK), α-SMA (1:400, MA5-14084; Thermo Fisher Scientific, Waltham, MA, USA), podocin (1:800, PA5-79757; Thermo Fisher Scientific, Waltham, MA, USA), synaptopodin (1:1,000, ab259976; Abcam, Cambridge, UK), p-p65 (1:800, MA5-15160), NLRP3 (1:800, ab263899), cleaved caspase-1 (1:1,000, PA5-99390; Thermo Fisher Scientific, Waltham, MA, USA), GSDMD (1:900, ab209845; Abcam, Cambridge, UK), and β-actin (1:8,000, MA1-140; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. After being washed three times with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1:7,000) for 1 h, and then the immunoblots were visualized using an ECL kit (Roche, Basel, Switzerland) and quantitated using ImageJ software (Bethesda, Rockville, MD, USA).
Ab182858
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab182858 is an antibody product from Abcam. It is a recombinant monoclonal antibody.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (142 mentions)
Pvdf membrane, by Merck Group (47 mentions)
Ab182733, by Abcam (27 mentions)
Ab9485, by Abcam (25 mentions)
Ab8245, by Abcam (24 mentions)
Ab32042, by Abcam (23 mentions)
Ab184787, by Abcam (22 mentions)
Ab2302, by Abcam (21 mentions)
Ab205718, by Abcam (21 mentions)
Ab181602, by Abcam (17 mentions)
Ripa buffer, by Beyotime (15 mentions)
Ripa lysis buffer, by Beyotime (14 mentions)
Ab13847, by Abcam (14 mentions)
Ab8227, by Abcam (14 mentions)
Ab8226, by Abcam (14 mentions)
Ab32351, by Abcam (13 mentions)
Ab6721, by Abcam (13 mentions)
Bca protein assay kit, by Beyotime (11 mentions)
Ab32536, by Abcam (10 mentions)
Ab214430, by Abcam (10 mentions)
256 protocols using ab182858
1
Quantitative Analysis of Apoptosis and Inflammation Markers
2
Extraction and Characterization of MO-B from Ophiopogon japonicus
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Ophiopogon japonicus was obtained from farms in Cixi (Zhejiang, China). MO-B was extracted from Ophiopogon japonicus using high-speed counter-current chromatography (19 (link)) and the yield was ~0.2–0.4 mg/g in tuber roots of Ophiopogon japonicus. High-performance liquid chromatography (HPLC) was conducted to measure the purity of MO-B, which was >97% (Fig. S1 ).HPLC was performed using a Shimadzu C18 column (5 µm 250×4.6 mm). The volume ratio of mobile solvents A (water) and B (acetonitrile) was maintained at 35:65, and the temperature was set at 30°C. The flow rate of the mobile phase was 1 ml/min. The detection wavelength was 285 nm. MO-B was then dissolved to 10, 20, 40 and 50 µM in dimethyl sulfoxide for cell treatment.
Antibodies targeting Bax (ab182733), Bcl-2 (ab182858), cleaved caspase-3 (ab32042), neutrophil cytochrome b light chain (p22phox; ab80896) and GAPDH (ab9482), goat anti-mouse horseradish peroxidase IgG (ab6789) and goat anti-rabbit IgG horseradish peroxidase (ab6721) secondary antibodies, were purchased from Abcam. Cell Counting Kit-8 (CCK-8), ROS and malondialdehyde (MDA) detection kits, radioimmunoprecipitation assay (RIPA) lysis buffer, a BCA Protein Assay kit and superoxide dismutase (SOD) assay kit with WST-8 were purchased from Beyotime Institute of Biotechnology.
Antibodies targeting Bax (ab182733), Bcl-2 (ab182858), cleaved caspase-3 (ab32042), neutrophil cytochrome b light chain (p22phox; ab80896) and GAPDH (ab9482), goat anti-mouse horseradish peroxidase IgG (ab6789) and goat anti-rabbit IgG horseradish peroxidase (ab6721) secondary antibodies, were purchased from Abcam. Cell Counting Kit-8 (CCK-8), ROS and malondialdehyde (MDA) detection kits, radioimmunoprecipitation assay (RIPA) lysis buffer, a BCA Protein Assay kit and superoxide dismutase (SOD) assay kit with WST-8 were purchased from Beyotime Institute of Biotechnology.
3
Thymoquinone-Induced Apoptosis and Autophagy
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Thymoquinone (TQ) was product of MCE (CAS: 490-91-5). Hoechst 33342 (B2261) was bought from Sigma-Aldrich. Cell counting kit‐8 (CCK‐8) was product of Selleck Chemicals (US). N-Acetyl-L-cysteine (NAC) and Z‐VAD‐FMK (ZVF) were obtained from Beyotime Biotechnology (Shanghai, China). The Apoptosis Detection Kit #556547 was purchased from BD (San Jose, CA). Antibodies against Bax (ab32503), Bcl‐2 (ab182858), LC3B (ab192890), Beclin-1 (ab207612), ATG7 (ab133528), MMP2 (ab92536), MMP9 (ab76003) and PD-L1 (ab213524) were bought from Abcam (San Francisco, CA). Antibody against vimentin (V6389) was purchased from Sigma-Aldrich (US). Antibodies against β‐actin (20536‐1‐AP) and Bcl-xl (10783-1-AP) were obtained from Proteintech (Chicago, IL). Antibodies against cleaved caspase 3 (9664), cleaved poly (ADP‐ribose) polymerase (PARP) (5625), E-cadherin (3195), N-cadherin (13116), and SQSTM1/p62 (8025) were obtained from CST company (NJ, US).
4
Western Blot Analysis of Apoptosis Markers
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Total protein was extracted from cells and uterine tissue with a protein extraction kit (KeyGEN, Changchun, China) according to protocols from the supplier. Protein concentration was determined using a bicinchoninic acid assay (KeyGEN). Equal concentrations of total protein were separated by SDS-PAGE (12%). Proteins were transferred onto polyvinylidene difluoride membranes and were blocked for two hours with TBST (50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20) containing 5% BSA. The membranes were incubated with primary antibodies diluted in TBST overnight at 4 °C. Antibodies against cytochrome C (ab133504), caspase-3 (ab184787), BAX, (ab32503), and Bcl-2 (ab182858) were from Abcam (Shanghai, China). After washing three times in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for two hours at room temperature and then washed three times for 10 min. Protein bands were visualized by exposure to an enhanced chemiluminescence detection system imager (Tanon Biotech, Shanghai, China) with an enhanced chemiluminescence solution (DiNING, Beijing, China). The relative intensity of each band was assessed by Image J 1.47 v software.
5
Quantitative Analysis of Apoptosis Regulators
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A2780 cells in each group were split by RIPA reagent (Yeasen, Shanghai, China) for 30 min on ice. Equal amounts of samples (20 μg) were separated by 10% SDS-PAGE and then electrophoretically shifted onto PVDF membranes, followed by blocking with 5% skim milk powder for 2 h. The membranes were interacted with primary antibodies against B-cell lymphoma-2 (Bcl-2) (1 : 2000, ab182858, Abcam, Cambridge, MA, USA) and BCL2-associated X (Bax) (1 : 1000, ab32503, Abcam) and GAPDH (1 : 5000 m ab8245, Abcam) all night at 4°C. Following secondary incubation at 37°C for 1 h, protein signals were quantified by an ECL reagent (Beyotime).
6
Immunohistochemical Analysis of Bcl2 in Liver
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Liver tissue samples were fixed in neutral buffered formalin (10%). Fixed samples were processed and stained with hematoxylin and eosin as described by Bancroft et al. (97 ). The immunohistochemical protocol was conducted following the method of Saber et al. (98 (link)). Wax was removed from tissue sections, and they were rinsed in 0.05 M citrate buffer, pH 6.8. Non-specific binding was blocked by treating the sections with 0.3% H2O2 and a protein block. Sections were subjected to rabbit monoclonal (anti-bcl2, Abcam, Cat# ab182858, dilution 1:500) primary antibody. Sections were washed in phosphate-buffered saline and subjected to goat antirabbit secondary antibody (EnVision System Horseradish Peroxidase Labeled Polymer; Dako) for 30 min at room temperature. Slides were visualized with DAB kit and counterstained using Mayer's hematoxylin. The immunolabeling indices of Bcl2 are presented as a percentage of positive expression in a total of 1,000 cells per eight high power fields (HPF).
7
Quantifying Protein Expression via Western Blot
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The total protein was extracted using a radioimmunoprecipitation assay, and the bicinchoninic acid (Beyotime, Shanghai, China) method was employed to quantify the concentration. The protein sample was first electrophoresed for 2h; subsequently, the authors transferred the total protein onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). TBST containing 5% skim milk was used to block non-specific antigens for 1h following incubating with primary antibodies (caspase-3 [1:1,000, ab32351; Abcam], c-caspase-3 [1:1,000, ab32042; Abcam], CREB1 [1:1,000, ab32515; Abcam], p-CREB1 [1:1,000, ab32096; Abcam], Bcl-2 [1:1,000, ab182858; Abcam], PSD-95 [1:1,000, ab238135; Abcam], synaptophysin [1:1,000, ab32127; Abcam], synapsin [1:1,000, ab254349; Abcam], GAPDH [1:1,000, ab8245; Abcam], and β-actin [1:5,000, #A5441, Sigma]) at 4°C overnight. Membranes were washed with TBST three times and incubated with the secondary HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution; cat. ab205719; Abcam). Subsequently, membranes were washed with TBST three times. Blots were then visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, Little Chalfont, UK).
8
Protein Expression Analysis in Tissue/Cells
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Sirt6, Nephrin, Desmin, CD86, CD206 proteins were extracted from the tissues or cells using RIPA lysis buffer (Beyotime, Shanghai, China). The concentrations of proteins were detected using the BCA protein kit (Beyotime, Shanghai, China). Aliquot protein was separated by 12% SDS-PAGE and resolved proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), which were blocked in 5% skim milk PBS with 0.1% Triton X-100 and incubated with primary antibodies as follows: anti-Nephrin antibody (1:1,000; ab216341), anti-Desmin antibody (1:1,000; ab15200), anti-Sirt6 antibody (1:2,000; ab191385), anti-CD86 antibody (1:5,000; ab53004 and ab112490), anti-CD206 antibody (1:1,000; ab64693), anti-Bcl-2 antibody (1:2,000; ab182858) and anti-Bax antibody (1:1,000; ab32503) (all from Abcam) overnight at 4°C. The membranes were then incubated with the appropriate HRP-conjugated secondary antibody (Proteintech Group, Inc./Thermo Fisher Scientific, SA00001-2, 1:5,000). Protein bands were detected with ECL (Thermo Fisher Scientific) and visualized using Quantity One software (Bio-Rad, Hercules, CA, USA).
9
Western Blot Analysis of Protein Expression
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The treated or untreated cells were collected, and the total protein was extracted by RIPA reagent (P0013B, Beyotime) with 1% protease inhibitors (P1030, Beyotime). After that, the concentration of the protein was determined by BCA kit (P0012, Beyotime). Then, cellular protein of 30 μg was separated by SDS-PAGE gel (P0012A, Beyotime). After electrophoresis for 90 min, the proteins were transferred on PVDF membranes (ISEQ00010/IPVH00010, MILLIPORE, MA, USA). Then, the membranes were blocked by 5% skimmed milk (D8340, Solarbio) for 1 h at room temperature, followed by incubation in primary antibodies (RUNX2, ab23981, 1：1,000, 60 kDa, Abcam, Cambridge, MA, USA; OCN, ab93876, 1：500, 11 kDa, Abcam; BCL2, ab182858, 1：2,000, 26 kDa, Abcam; GAPDH, ab181602, 1：10,000, 36 kDa, Abcam) at 4℃ overnight. Next day, the primary antibodies were recycled, and TBST (ST-673, Beyotime) was used to wash the membranes for 4 times (5 min for each time). Afterwards, secondary antibody (Goat Anti-Rabbit IgG H&L (HRP): ab6721, 1：10,000, Abcam) was incubated the membranes for 1 h (room temperature), followed by washing with TBST for 6 times (5 min for each time). Finally, ECL solution (WBKLS0500, MILLIPORE, Billerica, MA, USA) was dripped onto the membrane, and a specific imaging system (Bio-Rad, CA, USA) was used to visualize the band.
10
Mitochondrial Dynamics and Apoptosis Regulation in Liver Cells
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The liver tissue and HepG2 cells were lysed by RIPA lysis solution (Beyotime, China) to extract the protein. The protein concentration was detected by BCA reaction kit. After quantitative analysis, the total protein was denatured in this study. SDS-Page gel was used for electrophoresis, electrophoresis apparatus (Bio-RAD, USA) was adjusted to 120 V for electrophoresis, PVDF membrane (Millipore, USA) was used for membrane transfer, and skim milk (Sigma, USA) for blocking. Primary antibodies (Abcam, UK): Sirt1 (1:1000; ab189494), optic atrophy 1 (Opa1, 1:1000; ab157457), mitofusin 2 (Mfn2, 1:1000; ab124773), Drp1 (1:1000; ab184247), NRF1 (1:1000; ab34682), mitochondrial transcription factor A (TFAM, 1:1000; ab252432; Abcam; UK), Bcl-2 (1:2000; ab182858), cleaved-caspase 3 (1:500; ab2302), BCL2-associated X protein (Bax, 1:1000; ab32503), cleaved-caspase 9 (1:2000; ab32539) and GAPDH (1:2500; ab9485) were then added overnight to incubate. The next day, goat anti-rabbit antibody (1:2000; ab288151) was incubated for 1 h with slow shaking at 25 °C. The immunoreactive bands were visualized by intensive chemiluminescent reagent. The gray value was analyzed by ImageJ software.
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