Total protein and nucleoprotein (for Nrf2 and β-TrCP) in the ipsilateral peri-infarct hemisphere were extracted, and the protein concentration was determined by bicinchoninic acid protein (BCA) assay. The protein samples were then denatured by boiling for 10 min. Then, 20 μg of each protein sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V for 100 min. After transfer to polyvinylidene fluoride (PVDF) membrane, the separated protein bands were incubated with individual primary antibodies (caspase-3 1:500, ab197202, Abcam; AKT, 1:1000, Cat# 9272, CST; p-AKT, 1:1000, Cat#9271, CST; GSK-3β, 1:1000, Cat# 9315, CST; p-GSK-3β, 1:1000, Cat# 9336, CST; β-TrCP, 1:1000, Cat# 4394, CST; Nrf2 1:1000, ab92946, Abcam; PCNA, 1:1000, ab92552, Abcam; HO-1, 1:2000, ab52947, Abcam; NQO1, 1:10000, ab80588, Abcam) at 4 °C overnight, followed by incubation with corresponding secondary antibodies at room temperature for 2 h. The developed bands were observed under a gel imaging system.
Ab197202
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab197202 is a laboratory equipment product. It is designed for use in scientific research and experiments. The core function of this product is to facilitate specific laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (3 mentions)
Ab80588, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
Ab92946, by Abcam (1 mentions)
Ab52947, by Abcam (1 mentions)
Annexin 5 pi kit, by Keygen Biotech (1 mentions)
Pifithrin α, by Apexbio (1 mentions)
Ab32064, by Abcam (1 mentions)
Ab74290, by Abcam (1 mentions)
Ab84903, by Abcam (1 mentions)
Ab69540, by Abcam (1 mentions)
Ab2323, by Abcam (1 mentions)
Ab76424, by Abcam (1 mentions)
Rpmi 1640 culture medium, by Sartorius (1 mentions)
Ab2302, by Abcam (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Ecl prime kit, by GE Healthcare (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
14 protocols using ab197202
1
Quantification of Nrf2 and β-TrCP Proteins
2
Colorectal Cancer Apoptosis Regulation
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The tested compounds were provided by Dongdong Sun (Nanjing University of Chinese Medicine). For the treatment, the compounds were dissolved in culture medium and DMSO [with DMSO less than 0.1%(v/v)]. HCT116 colorectal cancer cells were supplied from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 culture medium was provided by Biological Industries (Beit‐Haemek). TUNEL apoptotic commercial kit was produced by Beyotime. Annexin V‐PI kit was purchased from KeyGEN. α‐ketoglutarate as obtained from Jinglai Biotechnology. The commercial kits for ATP, glutamine, glutamate and glutathione were provided by Jiancheng Biotechnology. The ASCT2 inhibitor GPNA was produced by Sigma‐Aldrich. Pifithrin‐α, the selective inhibitor for P53, was obtained from APExBIO. ASCT2 (#ab84903, 1:1000), P53 (#ab26, 1:1000), caspase‐3 (#ab197202, 1:1000), caspase‐7 (#ab69540, 1:1000), cleaved‐caspase‐3 (#ab2302, 1:1000), cleaved‐caspase‐7 (#ab2323, 1:1000), cleaved‐PARP (#ab32064, 1:1000), PARP (ab74290, 1:1000) and survivin (ab76424) were purchased from Abcam.
3
Western Blot Analysis of Apoptosis Markers
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For western blotting, 20 mg of protein was applied to the lanes of 4% to 12% Bis-Tris Gels (Life Technologies), then blotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA), and incubated with the relevant primary antibodies such as anti-Bax antibody [E63] ab32503 and anti-caspase-3 antibody [E87] ab197202 (Abcam, Cambridge CB2 0AX UK). Appropriate species-specific conjugated secondary antibody goat anti-rabbit HRP (ab205718) was commercially obtained (Abcam, Cambridge CB2 0AX UK). Proteins were detected using the ECL prime kit or the ECL kit (GE Healthcare Tokyo Japan) with an Image Quant LAS 4000 system (GE Healthcare). All protein expression levels were normalized to the levels of GPDH protein expression in each band.
4
Protein Expression Analysis Methods
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Primary antibodies used for Western blot, immunohistochemistry and immunocytochemistry are listed as followed: Hif-1α (79233, Cell signaling, USA), α-SMA (ab32575, Abcam, USA), Bnip3 (ab10433, Abcam, USA; ab38621, Abcam, USA), LC3B (83506, cell signaling, USA; 12741, cell signaling, USA), vimentin (ServiceBio, GB12192, China), phosphor-vimentin Ser56 (ab217673, Abcam, USA), phosphor-vimentin Ser38 (ab52942, Abcam, USA), phosphor-vimentin Ser72 (ab52944, Abcam, USA), phosphor-vimentin Ser82 (ab52943, Abcam, USA), caspase 3 (ab197202, Abcam, USA), cytochrome C (sc-13156, Santa Cruz, USA), GAPDH (GB12002, Servicebio, China).
5
Quantifying STRADA and Caspase-3 Protein Expression
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Western blot was performed to detect STRADA and caspase-3 protein expression, as previously described [24 (link)]. Briefly, the cells were harvested and lysed in cold RIPA cell lysis buffer (Beyotime Biotechnology, Inc., Shanghai, China) for 20 min. The mixture was then centrifuged to collect the supernatant containing the total protein. A BCA Protein Assay Kit (Beyotime Biotechnology, Inc., Shanghai, China) was used to measure the concentration of total protein. A total of 20 μg of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and incubated at 4°C overnight with the primary antibodies to STRADA (1: 1000) (ab230118; Abcam, Cambridge, MA, USA), caspase-3 (1: 1000) (ab197202; Abcam, Cambridge, MA, USA), and GAPDH (1: 1000) (ab9485; Abcam, Cambridge, MA, USA). The membranes were incubated with the secondary anti-mouse or anti-rabbit antibody as 1: 5000 dilution for 90 min (Abcam, Cambridge, MA, USA). The ECL Substrate Kit (Abcam, Cambridge, MA, USA) was used to visualize the protein bands.
6
Investigating P-gp Expression in Doxorubicin-Treated K562 Cells
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To investigate the expression of P-gp levels in K562 cells treated with various formulations of DOX (10 μg/mL), western blotting was performed. Briefly, total protein lysates were collected in a lysis solution containing 1% PMSF. And total protein concentration was measured using a BCA protein assay kit (CWBIO, Beijing, China). Equal amounts of protein from each sample were separated using 10% SDS-PAGE gels before transferring to a polyvinylidene difluoride membrane. Membranes were blocked in nonfat milk and then incubated with primary antibodies against P-gp and β-actin (Santa Cruz Biotech, #sc-47778) overnight at 4°C. The next day, blots were incubated with a secondary antibody at 37°C for 2 h. Bands were visualized using ECL according to the manufacturer’s protocol.
Antibodies against Bim (ab32158), Bad (ab32445), Bax (ab32503), caspase 3 (ab197202) and caspase 9 (ab219590) were purchased from Abcam. Equal loading between samples was determined analyzed by β-actin expression levels.
Antibodies against Bim (ab32158), Bad (ab32445), Bax (ab32503), caspase 3 (ab197202) and caspase 9 (ab219590) were purchased from Abcam. Equal loading between samples was determined analyzed by β-actin expression levels.
7
Thyroid Adenoma and Hashimoto's Thyroiditis
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Following informed consent of the patients, another 20 cases of thyroid adenoma with Hashimoto's thyroiditis were collected as the case group, and 20 cases of normal tissues adjacent to benign thyroid adenoma were collected as the control group. Nthy-ori3-1 cells (normal cells of human thyroid follicular epithelium) were purchased from the Wuhan Cell Bank of the Chinese Academy of Sciences. The rabbit anti-β-actin antibody (ab179467), rabbit anti-LC-3 antibody (ab48394), rabbit anti-mTOR antibody (ab109268) and rabbit anti-caspase-3 antibody (ab197202) were all purchased from Abcam. RPMI-1640 culture medium was purchased from BD Biosciences. Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, Inc.). The MTT cell proliferation kit (cat. no. 4890-025-k) was purchased from Trevigen, USA. 25-(OH)D3 was purchased from Shanghai Yubo Biotechnology Co., Ltd. (D455087). Double-distilled water (ddH2O) was used to prepare 20, 40, 60, 80 and 100 mmol/l solutions.
8
Protein Extraction and Western Blot Analysis
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After 72-h transfection, cells were collected, washed twice with pre-cooled PBS, and lysed on ice with buffer containing 10 µl phosphatase inhibitor, 1 µl protease inhibitor, and 5 µl 100 mM PMSE for 30 min. The resulting cell lysate was centrifuged at 14,000 rpm for 15 min at 4℃, and the supernatant was aspirated for protein quantification by the Bradford method. Proteins were separated by SDS-PAGE electrophoresis, electro-transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking at room temperature for 2 h, membrane was incubated with primary antibody overnight at 4℃, and then probed with secondary antibody at room temperature for 1–2 h. The blot was visualized by G: BOX chemiXR5 imaging and analyzed by Gel-Pro32 software. The primary antibodies against CDC6 (ab109315), caspase-3 (ab197202), bcl-2 (ab182858), Bax (ab182734), E-cadherin (ab40772), and ATR (ab13798) were purchased from abcam, UK; anti-INK4 (Cst 74,560) was from Cell Signaling Technology, US.
9
Western Blot Analysis of Cellular Markers
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Cells were lysed in lysis buffer with protease inhibitor, and equal amounts of lysates were separated by SDS-PAGE and transferred onto the membranes, which were incubated with antibodies for SATB1 (Abcam, UK, #ab109122), MMP-2 (Abcam, UK, #ab92536), Vimentin (Abcam, UK, #ab137321), E-Cadherin (Abcam, UK, # ab201499), Bcl-2 (Abcam, UK, #ab194583), caspase-3 (Abcam, UK, #ab197202), caspase-8 (Abcam, UK, #ab108333), GAPDH (Abcam, UK, #ab37168). The blots were washed, incubated with secondary antibodies (Abcam, UK), and developed using enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA).
10
Immunohistochemical Analysis of SIRT1 and Cleaved Caspase-3 in Spinal Cord Tissue
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After paraffin embedding and sectioning (4 μm), the peripheral spinal cord tissues around T9–10 at the dorsal part (weight: 1 g, volume: 2 ml) were dewaxed with xylene, hydrated with gradient alcohol, and inactivated with 3% H2O2 for 10 min. Microwave repair (pH = 6.0, 15 min) was performed with 0.01 mol/L sodium citrate buffer solution. After being blocked with 5% bovine serum albumin (BSA) for 20 min, the sections were incubated with primary antibodies of SIRT1 (Abcam, ab110304, 1:1000, MA, United States) and Cleaved caspase-3 (Abcam, ab197202, dilution concentration 1:100, Ma, United States) overnight at 4°C. The next day, the secondary antibodies were added respectively and incubated at room temperature for 20 min. DAB color was developed after PBS washing. After counterstaining with hematoxylin, the sections were dehydrated and mount examined under a microscope.
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