Immunoblotting experiments were performed as previously described [39 (link)]. The following primary antibodies were used: 1:1000 ANTI-Beclin 1 (Abcam, Cambridge, UK), 1:10000 ANTI-APG7 (Santa Cruz, Dallas, TX, USA), 1:500 ANTI-LC3A/B (Abcam, Cambridge, UK), 1:5000 ANTI-β-actin (Sigma, Saint-Louis, MO, USA). Images were recorded by the ImageQuant Las 500 (GE Healthcare, Chicago, IL, USA) and analyzed using Image Lite Studio software (LI-COR, Lincoln, NE, USA).
Anti lc3a b
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-LC3A/B is a primary antibody that recognizes the mammalian homologs of yeast Atg8, LC3A and LC3B. It can be used to detect these autophagy-related proteins in various species and applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti beclin1, by Abcam (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti ve cadherin, by Abcam (2 mentions)
Anti zo 1, by Abcam (2 mentions)
Anti smac, by Abcam (2 mentions)
Anti cleaved caspase 9, by Abcam (2 mentions)
Ripa buffer, by Wuhan Servicebio Technology (2 mentions)
Anti caspase 9, by Abcam (2 mentions)
Anti caspase 3, by Abcam (2 mentions)
Anti occludin, by Abcam (2 mentions)
0.45 μm pvdf membrane, by Merck Group (2 mentions)
Except for labeled, by Cell Signaling Technology (2 mentions)
Anti errα, by Abcam (2 mentions)
Anti jam a, by Abcam (2 mentions)
Anti sirt3, by Abcam (2 mentions)
Anti p62, by Abcam (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
Anti bax, by Abcam (2 mentions)
Anti cytochrome c, by Abcam (2 mentions)
Image studio lite software, by LI COR (1 mentions)
10 protocols using anti lc3a b
1
Immunoblotting for Autophagy Markers
2
Comprehensive Lung Protein Analysis
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The total protein of lung tissues and cells was collected with RIPA buffer (Servicebio, Wuhan, China) with cocktail and phosphatase inhibitors (Thermo Fisher Scientific, Wuhan, China), followed by electrophoresed through 8–12% sodium dodecyl sulfate–polyacrylamide gel and then transferred to 0.45 μm PVDF membrane (Merck Millipore, USA). A total of 30 ug of protein was used for western blot experiments. After blocked with 5% milk, the membrane was immunoblotted with following primary antibodies(Except for labeled, all from Cell Signaling Technology, MA, USA) at 4 °C overnight: anti-ERRα (1:1000), anti-ZO-1 (1:1000; Abcam), anti-VE-cadherin (1:1000), anti-Occludin(1:1000), anti-JAM-A(1:1000; Abcam), anti-Sirt3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-Smac (1:1000), anti-Cytochrome c (1:1000), anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:500), anti-caspase 9 (1:1000), anti-cleaved caspase 9 (1:500), anti-LC3A/B(1:1000), anti-Beclin1 (1:1000), anti-p62 (1:1000), or anti-GAPDH (1:10000, Abcam, internal control). After incubated with the HRP-conjugated secondary antibodies at 25℃ for 1 h, the immunoblots were visualized using the ECL system. The gray value was analyzed using the Image J and calculate the relative protein level based on the density ratio of the target protein to GAPDH (internal control).
3
Autophagy Induction and LC3 Analysis
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The microtubule-associated protein 1 light-chain 3 (LC3) is essential for amino-acid starvation-induced autophagy, and it is associated with the autophagosome membrane.26 (link)–28 (link) RG2 cells (1×106) were cultured on six-well plates. After treatment, the cells were fixed in 4% paraformaldehyde, blocked with 3% normal goat serum and incubated in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h to permeabilize the cells and to block nonspecific protein–protein interactions. Afterwards, cells were incubated with the goat polyclonal anti-LC3A/B (Abcam, Cambridge, UK) for 30 min. Then, cells were washed twice with PBS and incubated for additional 30 min in darkness with an anti-goat IgG antibody coupled with APC (Abcam), washed again with PBS and finally acquired using a FACSCalibur flow cytometer using the CellQuest software and FlowJo 10× for the final analysis (Becton-Dickinson).
4
Baicalin Modulates Autophagy in Irradiated Cells
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CNE-2R cells were seeded overnight in a six-well plate (6 × 104 cells/well). The cells were pretreated with or without 10 μg/ml baicalin and 4 Gy X-ray dose. Protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Billerica, MA). The membranes were blocked at room temperature for 1 h in 5% skim milk in Tris-buffered saline with Tween 20 and then incubated overnight at 4 °C with the following indicated primary antibodies: anti-LC3A/B (Abcam, Cambridge, UK), BECN1 polyclonal (ABclonal, US), and GAPDH antibodies (Abcam, Cambridge, UK). Goat anti-rabbit IgG was used as the secondary antibody (Abcam, Cambridge, UK). Fluorophores were detected using the Infrared Fluorescence Imaging System (BioRad Universal Hood III).
5
Western Blot Analysis of Autophagy Markers
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Antibodies were obtained from commercial sources: anti-NMDAR1, anti-NMDAR2B, and anti-LC3A/B; anti-APG5 and anti-ULK1 (Abcam, Cambridge, UK); anti-beclin1, anti-AMP-activated protein kinase (AMPK) α, and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA).
For the western blot analysis, the cells were rinsed with phosphate-buffered saline (PBS) and subsequently lysed for 30 min on ice in RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM DTT, and 50 μg/mL PMSF). The insoluble material was removed by centrifugation at 12000 rpm for 20 min. Next, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were blocked in PBS with 5% skim milk and 0.05% Tween 20, incubated with the diluted primary antibodies against NMDAR1, NMDAR2B, or LC3A/B (1: 1000; Cell Signaling Technology, Denvers, MA, USA); APG5 or ULK1 (1: 1000; Abcam, Cambridge, UK); beclin-1 or AMPKα (1: 200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and followed by incubation with a horseradish peroxidase-conjugated secondary antibodies (1: 2500; Santa Cruz). β-actin (Santa Cruz) was used as a control. The blots were assayed using an enhanced chemiluminescence detection system (Image Quant LAS 4000mini, Pittsburgh, PA, USA).
For the western blot analysis, the cells were rinsed with phosphate-buffered saline (PBS) and subsequently lysed for 30 min on ice in RIPA-B buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 50 mM NaF, 100 μM Na3VO4, 1 mM DTT, and 50 μg/mL PMSF). The insoluble material was removed by centrifugation at 12000 rpm for 20 min. Next, the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blots were blocked in PBS with 5% skim milk and 0.05% Tween 20, incubated with the diluted primary antibodies against NMDAR1, NMDAR2B, or LC3A/B (1: 1000; Cell Signaling Technology, Denvers, MA, USA); APG5 or ULK1 (1: 1000; Abcam, Cambridge, UK); beclin-1 or AMPKα (1: 200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and followed by incubation with a horseradish peroxidase-conjugated secondary antibodies (1: 2500; Santa Cruz). β-actin (Santa Cruz) was used as a control. The blots were assayed using an enhanced chemiluminescence detection system (Image Quant LAS 4000mini, Pittsburgh, PA, USA).
6
Aloperine Modulation of Autophagy Signaling
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Cells were cultured in 10-cm cell culture dishes and treated with aloperine. DMSO was used as a negative control. The whole cellular extract was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the separated proteins were electrically transferred to a PVDF membrane (Millipore Corporation, USA). The membrane was blocked with primary antibodies (anti-LC3 Ab; Abcam, USA; anti-GAPDH Ab; GeneTex, USA; anti-AMPK α-1 Ab; Cell signaling, USA; anti-phosphorylated-AMPK α-1 (Thr 172) Ab; Cell Signaling, USA; anti-Akt Ab; Santa Cruz, USA; anti-phosphorylated-Akt (Ser 473) Ab; Santa Cruz, USA; anti-mTOR Ab; Cell Signaling, USA; anti-phosphorylated-mTOR (Ser 2448) Ab; Cell Signaling, USA; anti-p70S6K Ab; Cell Signaling, USA; anti-phosphorylated-p70S6K (Thr 389) Ab; Cell Signaling; anti-p62/SQSTM1 Ab; Abgent, USA; anti-Erk Ab; Cell Signaling, USA; anti-phosphorylated-Erk (Thr 202/204) Ab; Cell Signaling, USA; anti-JNK Ab; Cell Signaling, USA; anti-phosphorylated-JNK (Thr 183/Tyr 185) Ab; Cell Signaling, USA; anti-p38 Ab; Cell Signaling, USA; and anti-phosphorylated (Thr 180/Tyr 182) Ab; Cell Signaling Ab, USA) and was analyzed using the BioSpectrum 800 Imaging System (UVP, CA, USA).
7
Western Blot Analysis of Autophagy Markers
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Whole cell extracts were prepared using 0.14 M NaCl, 0.2 M triethanolamine, 0.2% sodium deoxycholate, 0.5% Nonidet P-40 and supplemented with a protease inhibitor (all of the products were from Sigma, St. Louis, Missouri, USA). Then, protein sample was run through a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a membrane. The transferred membranes were subsequently incubated overnight at 4°C with a primary antibody. After washing, the membrane was incubated with a horseradish peroxidase (HRP)-linked secondary antibody for 1 h at room temperature. The primary antibodies were anti-ATG-5 (Santa Cruz, CA, USA), anti-LC3A/B (abcam, Cambridge, UK) and anti- β-Actin (Santa Cruz, CA, USA). All reported results are the average ratios of three different independent experiments.
8
Western Blot Analysis of Protein Expression
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Western blotting was performed to detect the levels of thrombin, PAR-1, SIRT1, LC3I, LC3II, and Beclin1. Briefly, the total protein extracted from the sample was measured using a protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by sodium dodecyl sulfate (10%) polyacrylamide gel electrophoresis (10% SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked with 5% nonfat milk at room temperature for 1 h. The membranes were then incubated with primary antibody overnight at 4°C: anti-thrombin (ab92621, 1 μg/ml), anti-SIRT1 (ab189494, 1/1000), anti-LC3A/B (ab128025, 2 μg/ml), anti-Beclin 1 (ab210498, 1/1000), and anti-GAPDH (ab8245, 1/500) were all purchased from Abcam (Cambridge, MA, USA), while anti-PAR1 (#MBS3008648, 1/500) was purchased from MyBioSource, Inc. (San Diego, CA, USA). After washing 3 times with tris-buffered saline with Tween, the membranes were incubated with corresponding goat anti-mouse or goat anti-rabbit secondary antibodies for 1 h at room temperature. Protein bands were analyzed using the Bio-Image Analysis System (Bio-Rad, Richmond, CA, USA). The data were normalized to b-actin, which served as an internal control.
9
Flavonoid-Mediated Autophagy Modulation
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Flavonoid aglycons (except quercetin) were purchased from Extrasynthese (Genay Cedex, France) and were diluted in ethanol 100% to stock solutions 10 mg/mL, except for diosmetin that was diluted as 2 mg/mL stock due to its low solubility. Chloroquine (25 mM stock in PBS), palmitic acid (PA) (0.3 M stock in ethanol) and quercetin (25 mM stock in ethanol) were from (Merk, Sigma-Aldrich, Darmstadt, Germany). The compounds were kept in aliquots at −20 °C and thawed shortly before each treatment. The antibodies (Abs) used for Western blotting (WB) were polyclonal rabbit anti-LC3A/B (Abcam plc, Cambridge, UK, ab1280025), anti-p62 (MBL International, Woburn, MA, USA, PM045), anti-GAPDH, clone FL335 (Santa Cruz Biotech., Dallas, Texas, USA, sc-25778), anti-NQO2 (Proteintech, Manchester, UK), and monoclonal mouse anti-α-Tubulin (Merk, Sigma-Aldrich, T6074). Ab used to detect phosphorylated (ph)AMPK alpha 1 was anti-AMPK alpha1 (phospho-T183) + AMPK alpha2 (phospho-T172) antibody (Abcam, ab133448).
10
Immunoblotting Analysis of Lung Proteins
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The total protein of lung tissues and cells was collected with RIPA buffer (Servicebio, Wuhan, China) with cocktail and phosphatase inhibitors (Thermo Fisher Scienti c, Wuhan, China), followed by electrophoresed through 8-12% sodium dodecyl sulfate-polyacrylamide gel and then transferred to 0.45μm PVDF membrane (Merck Millipore, USA). After blocked with 5% milk, the membrane was immunoblotted with following primary antibodies(Except for labeled, all from Cell Signaling Technology, MA, USA) at 4 °C overnight: anti-ERRα (1:1000), anti-ZO-1 (1:1000; Abcam), anti-VE-cadherin (1:1000), anti-Occludin(1:1000), anti-JAM-A(1:1000; Abcam), anti-Sirt3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-Smac (1:1000), anti-Cytochrome c (1:1000), anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:500), anti-caspase 9 (1:1000), anti-cleaved caspase 9 (1:500), anti-LC3A/B(1:1000), anti-Beclin1 (1:1000), anti-p62 (1:1000), or anti-GAPDH (1:10000, Abcam, internal control). After incubated with the HRP-conjugated secondary antibodies at 25℃ for 1h, the immunoblots were visualized using the ECL system. The gray value was analyzed using the Image J and calculate the relative protein level based on the density ratio of the target protein to GAPDH (internal control).
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