Western blot analysis was performed as previously described by our group (Cai et al., 2009). Cells were lysed and sonicated in lysis buffer. After electrophoresis, samples were transferred onto a polyvinylidene difluoride membrane (Millipore, Temecula, CA, USA), and then immunoblotted with the following antibodies: goat polyclonal anti-SIAH-1 (1:100; sc-5505; Santa Cruz Biotechnology), rabbit polyclonal anti-α-synuclein (1:1,000; 2642; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-light chain 3 (LC3) (1:1,000; ab62721; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-E1 (1:1,000; 4891S; Cell Signaling Technology), rabbit polyclonal anti-P53 (1:1,000; 2642; Cell Signaling Technology), and mouse monoclonal β-actin (1:1,000; A3854; Sigma-Aldrich, St Louis, MO, USA). Subsequently, the following horseradish peroxidase-conjugated secondary antibodies were added: polyclonal goat anti-rabbit secondary antibody for LC3, α-synuclein and E1; polyclonal donkey anti-goat secondary antibody for SIAH-1; and polyclonal goat anti-mouse secondary antibody for β-actin (1:1,000; Beyotime Biotechnology, Jiangsu, China). Images were captured using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and band intensities were calculated by densitometric analysis using Image J software (NIH, Bethesda, MD, USA).
Ab62721
Manufactured by Abcam
Sourced in United States
Ab62721 is an anti-Glucose-6-Phosphate Dehydrogenase antibody produced in rabbit and used for the detection of Glucose-6-Phosphate Dehydrogenase in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab207612, by Abcam (2 mentions)
Ab13847, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab32124, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti α synuclein, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
A3854, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab179515, by Abcam (1 mentions)
Ab215203, by Abcam (1 mentions)
Ab283684, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab98830, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab263899, by Abcam (1 mentions)
Ab240635, by Abcam (1 mentions)
Goat anti rabbit and anti mouse igg hrp antibodies, by Bioworld Technology (1 mentions)
Primary antibody against gapdh 60004 1 ig, by Proteintech (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
9 protocols using ab62721
1
Western Blot Analysis of Cellular Proteins
2
Quantification of Autophagy and Inflammasome Markers
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After transfection, cells were collected and lysed. Then, total protein was collected and concentrated using a BCA kit. Protein (30 µg) was isolated using 10% SDS-PAGE at 120v. The protein was transferred onto PVDF membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies such as antiLC3I/II (ab62721, 1:2000, Abcam, USA), antiATG8 (ab98830, 1:2000, Abcam, USA), antiNLRP3 (ab263899, 1:1000, Abcam, USA), antiASC (ab283684, 1:1000, Abcam, USA), anti-Caspase1 (ab179515, 1:1000, Abcam, USA), GSDMD (ab215203, 1:1000, Abcam, USA), antiβ-actin (ab8227, 1:5000, Abcam, USA) and goat-anti-rabbit antibody (ab6721, 1:5000, Abcam, USA). Finally, the bands were captured by ECL reagents and analyzed using ImageJ (V.2.3.0).
3
Streptozotocin-Induced Diabetes Model
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Glucose, streptozotocin (STZ), citrate buffer, dimethylsulphoxide (DMSO) and collagenase type II were purchased from Sigma‐Aldrich. Rapamycin was acquired from MedChemExpress LLC. Cell culture reagents were obtained from Gibco. Cell counting kit‐8 (CCK‐8) was purchased from Dojindo. Glucometer was obtained from Yuwell. The primary antibody against GAPDH (60004‐1‐Ig) was purchased from Proteintech, primary antibodies against p21WAF1 (ab86696), p62 (ab240635), LC3 (ab62721), and Atg7 (ab223365) were acquired from Abcam, and primary antibodies against HuR (#12582), p53 (#2524), p‐p53 (#9284), p16INK4A (#80772) and LC3‐II (#2775) were purchased from Cell Signaling Technology. Goat anti‐rabbit and anti‐mouse IgG‐HRP antibodies were purchased from Bioworld. In Jackson ImmunoResearch, Alexa Fluor® 594‐ and 488‐conjugated secondary antibodies were obtained. In Beyotime, 4′,6‐Diamidino‐2‐phenylindole (DAPI) was purchased.
4
Quantitative Analysis of Autophagy Markers
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Total protein was extracted using a kit (KGP250; Nanjing Keygen Biotech Co. Ltd., Nanjing, China). Whole cell lysat was separated by 10-15% SDS-PAGE then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder in TBST (0.1% Tween 20 in TBS) for 1 hr at room temperature and incubated overnight at 4°C with antibodies against LC3II (1:500; Abcam, ab62721), Beclin1(1:1000; Abcam, ab55878), P62 (1:1000; Abcam, ab91526), DUSP5 (1:400; Abcam, Cat# ab54939 ), ERK1/2 (1:700; CST, Cat# 4695), p-ERK1/2 (1:1000; CST, Cat# 4370) and GAPDH (1:2000; Santa Cruz Biotechnology; Santa Cruz, CA, USA), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:3000; Proteintech Group, Inc., Hubei, China). Immunoreactive bands were visualized using a chemiluminescence kit (ECL kit; Santa Cruz Biotechnology, USA), and protein bands were scanned using Chemi Imager 5500 V2.03 software. The integrated density value (IDV) for each band was calculated with a computer-aided image analysis system (Fluor Chen 2.0). The IDV of LC3II was normalized with the IDV of LC3I, while the other proteins were normalized with the IDV of GAPDH.
5
Protein Expression Analysis in Cells
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RIPA buffer lysed the cells to obtain the total protein and a BCA kit was used to quantify the total protein concentration in each group of cells. We separated 30 μg proteins with SDS-PAGE and transferred them to PVDF membranes with semi-dry transfer method. We sealed the PVDF membrane with 50 g/L skim milk powder for 2 h at room temperature. The PVDF membrane was incubated with primary antibody at 4°C overnight, and with secondary antibody at 37°C for 1 h. GAPDH was used as an internal control for the detection of XBP-1 (ab37152, Abcam), LC3I/II (ab62721, Abcam), GRP78 (ab21685, Abcam), ATF4 (ab184909, Abcam), ATF6 (ab203119, Abcam), IRE1 (ab37073, Abcam), CHOP (#2895, Cell Signaling Technology), XBP-1s (#27901, Cell Signaling Technology), XBP-1u (25997-1-AP, ProteinTech), Casp-4 (ab25898, Abcam), casp-3 (ab13847, Abcam), c-casp-3 (ab2302, Abcam), Bax (ab32503, Abcam), Bcl-2 (ab32124, Abcam), Beclin-1 (ab207612, Abcam) and Atg5 (#2630, Cell Signaling Technology). Then, ImageJ software was used for grayscale scanning and quantification.
6
Protein Analysis Using Western Blot
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Total protein was collected with the RIPA buffer (Beyotime). And the concentration of these proteins was measured with the BCA (Beyotime) method. Next, these proteins were separated with the 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime). After that, these proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). Then these membranes were blocked with the 5% skim milk powder and incubated with the primary antibodies at 4 °C overnight. The primary antibodies used in this research were BNIP3 (ab109362, ABCAm), Bcl-2 (ab32124, ABCAm), Bax (ab32503, ABCAm), Cleaved caspase3 (ab49822, ABCAm), Cleaved caspase9 (ab2324, ABCAm), Little Computer 3 (LC3) I/II (ab62721, ABCAm), Autophagy-related protein 7 (ATG7) (ab133528, ABCAm), Beclin1 (#3495S, Cell Signaling Technology, USA), P62 (#5114S, Cell Signaling Technology), and GAPDH (ab8245, ABCAm). On the second day, these membranes were washed with the phosphate-buffered solution (PBST) for three times and then incubated with the second antibody for 2 h at room temperature. At last, these membranes were washed with the PBST again and the immunoreactive signal was detected with the LAS-3000 Image Analyzer (Fujifilm, Tokyo, Japan).
7
Western Blot Analysis of Autophagy Regulators
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The cells were lysed in ice-cold whole cell extract buffer B (50 mM TRIS-HCl, pH 8.0, 4 M urea, and 1% Triton X-100) supplemented with protease inhibitor mixture. The cell extracts were resolved by SDS-PAGE and analyzed by western blotting. Protein bands were visualized using ECL Blotting Detection Reagents. All blots derive from the same experiment and they were processed in parallel. The antibodies used for western blotting include ATG3 (1:1000 dilution; 3415; Cell Signaling Technology), ATG5 (1:1000 dilution; 12994; Cell Signaling Technology), ATG7 (1:1000 dilution; 8558; Cell Signaling Technology), ATG9A (1:1000 dilution; 13509; Cell Signaling Technology), ATG16L1 (1:1000 dilution; 8089; Cell Signaling Technology), GCN5L2 (1:1000 dilution; 3305; Cell Signaling Technology), PCAF (1:1000 dilution; 3378; Cell Signaling Technology), P300 (1:1000 dilution; ab14984; Abcam), CBP (1:1000 dilution; ab253202; Abcam), SQSTM1 (1:1000 dilution; ab109012; Abcam), MAP1LC3A/B (1:1000 dilution; ab62721; Abcam), LAMP1 (1:1000 dilution; ab289548; Abcam), CTSB (1:1000 dilution; ab125067; Abcam), CTSD (1:1000 dilution; ab75811; Abcam), and β-Actin antibodies (1:1000 dilution; 60008-1-Ig; Proteintech).
8
Western Blot Analysis of Signaling Pathways
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Treated HUVECs or tissues were lysed by RIPA lysates (P0013B, Beyotime, Beijing). After being processed by the ultrasonic cell disruptor, the sample was centrifuged at 12,000°g at 4°C for 5°min on the ice. The supernatant was collected in a new EP tube. The BCA protein assay kit (#23227; Thermo, United States) was utilized to evaluate the protein concentration. The protein sample was separated by SDS-PAGE gel electrophoresis and transferred to the PVDF membrane. The membrane was sealed with 5% skimmed milk powder (232100-500°g, BD, United States) at room temperature for 2°h. Following being incubated with the primary antibodies against PI3K (1:1,000; ab32089; Abcam, United States), p-PI3K (1:1,000; ab86714; ab32089), Akt (4298; CST, United States), p-Akt (12694), mTOR (2972; Cell Signaling, United States), p-mTOR (2971; Cell Signaling), LC3-I / II (1:1,000; ab62721; Abcam), Beclin-1 (1:1,000; ab210498; Abcam), P62 (1:1,000; ab211324; Abcam), Bcl-2 (1:1,000; ab218123; Abcam), Bax (1:1,000; ab3191; Abcam), Caspase-3 (1:1,000; ab179517; Abcam) and β-actin (1:1,000; ab115777; Abcam) overnight at 4°C, samples were incubated with horseradish Peroxidase (HRP) labeled goat anti-rabbit (ZB-2301; ZSGB-BIO, Beijing, China) or anti-mouse (ZB-2305; ZSGB-BIO) IgG at room temperature for 2°h. The expression of target proteins was quantified by ImageJ software.
9
Western Blot Analysis of Apoptosis Markers
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The protein extracted from HK-2 cells were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes blocked with 5% non-fat milk at a room temperature for 3 h. Afterwards, the membranes were incubated with primary antibody at 4°C overnight, followed by horseradish peroxidase (HRP)conjugated secondary antibodies at 37°C for 1 h. The primary antibodies were as follows: Anti-SPRED2 (ab153700, 1/500), Anti-caspase-3 (ab13847, 1/500), Anti-BAX (ab182733, 1/2000), Anti-BCL-2 (ab194583, 1/500), Anti-LC3-II/I (ab62721, 1 µg/mL), and Anti-Beclin 1 (ab207612, 1/2000) (all purchased from Abcam). The GAPDH was used as a control. Protein bands were detected using Super Signal West Pico Chemiluminescent Substrate kit (NCM Biotech, Suzhou, China).
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