In addition to measuring LC-3 protein levels in freshly isolated endothelial cells, we also assessed autophagy status by counting the number of LC3-bound puncta, which reflects autophagosome formation. For HAEC’s, we counted the number of cells with more than twenty LC3-bound puncta per cell. Cells were stained with antibody against LC3B (1:100; Abcam, Cambridge, MA) as described above. An investigator blinded to subject status or cell culture condition counted puncta in twenty cells calculated the mean. To assess reproducibility, puncta count was performed twice in a blinded fashion (n=10). The coefficient of variation was 2.4%.
Antibody against lc3b
Manufactured by Abcam
Sourced in United Kingdom
Antibody against LC3B is a laboratory reagent used to detect the microtubule-associated protein 1 light chain 3 beta (LC3B) protein. LC3B is a widely used marker for monitoring autophagy, a cellular process involved in the degradation and recycling of cellular components. This antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of LC3B in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
P jnk, by Beyotime (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
P mtor, by Santa Cruz Biotechnology (3 mentions)
P akt, by Beyotime (3 mentions)
Snail, by Santa Cruz Biotechnology (3 mentions)
P erk, by Santa Cruz Biotechnology (3 mentions)
N cadherin, by Boster Bio (2 mentions)
Vimentin, by Boster Bio (2 mentions)
Beclin 1, by Boster Bio (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Sp600125, by Beyotime (2 mentions)
U0126, by Beyotime (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Ly294002, by Cayman Chemical (1 mentions)
Tetrahydrocurcumin, by Yuanye Bio-Technology (1 mentions)
6 protocols using antibody against lc3b
1
Quantifying Autophagy in Endothelial Cells
2
Immunofluorescence Analysis of Autophagic Markers
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MLTC-1 cells were fixed with PBS containing 4% paraformaldehyde for 1 h, and then permeabilized with 0.1% Triton X-100 for 30 min. After that, cells were blocked with 1% bovine serum albumin for 2 h, and then incubated overnight with an antibody against LC3B (1:100; 10 μg/mL, Abcam Cambridge, MA, USA) at 4°C. Later on, cells were incubated with horseradish peroxidase-conjugated secondary antibody (1:1000, Abcam) at room temperature for 1 h, and then observed under a confocal microscopy (Olympus CX23 Tokyo, Japan). The nucleus was stained with DAPI for 5 min. Autophagy inhibitor 3-Methyladenine (3MA) was purchased from Sigma Aldrich (St. Louis, MO, USA).
3
Investigating Apoptosis Signaling Pathways
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Antibodies against phospho-GSK-3β (Ser 9), GSK-3β, phospho-Stat3, Stat3, cleaved Caspase 3, and Survivin were from Cell Signaling Technologies (Danvers, MA). Antibodies against AIF and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against LC3B was from Abcam (Cambridge, MA). The antibody against SphK2 was from Exalpha Biologicals (Dublin, OH). The fluorescent labeling of inhibitors of caspases (FLICA) kit was from Immunochemical Technologies (Bloomington, MN). SphK inhibitor Ski-1 was from Biovision (Milpitas, CA). The pan caspase inhibitor Z-VAD was from Promega (Madison, WI). The PI3K inhibitor LY294002 was from Cayman (Ann Arbor, MI). And the Jak inhibitor JAKi was from Millipore (Billerica, MA). Human LC real time PCR arrays were from Origene (Rockville, MD).
4
Tetrahydrocurcumin Modulates Epithelial-Mesenchymal Transition
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Tetrahydrocurcumin was purchased from Yuanye Biotechnology Co. Ltd (Shanghai,China). E-cadherin, ZEB, Snail, Twist, HIF-1α, p-mTOR, mTOR, p-ERK, ERK, GAPDH and β-actin antibodies were purchased from Santa cruz Biotechnology (Santa cruz, CA, USA). Vimentin, N-cadherin, VEGF, MMP-2, MMP-9, LaminA/C, Atg5, Atg7 and Beclin-1 antibodies were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). LY294002, SB203580, Rapamycin, β-catenin, p-Akt, Akt, p-JNK, JNK, p-p38, p38, IκB-α, p65, Hsp90 and GSK-3β antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). Antibody against LC3B was purchased from Abcam (Cambridge, UK). All other reagents were from common commercial sources.
5
Andrographolide and Dihydroartemisinin Signaling
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Andrographolide and Dihydroartemisinin was purchased from Yuanye Biotechnology Co. Ltd (Shanghai,China). PI3K Class I, PI3K Class III E-cadherin, Snail, p-mTOR, mTOR, p-ERK, ERK and β-actin antibodies were purchased from Santa cruz Biotechnology (Santa cruz, CA, USA). Vimentin, N-cadherin, Atg5 and Beclin-1 antibodies were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). U0126, SP600125, p-Akt, Akt, p-JNK, JNK, p-p38 and p38 antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). Antibody against LC3B was purchased from Abcam (Cambridge, UK). All other reagents were from common commercial sources.
6
Western Blot Analysis of EMT Markers
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Proteins were extracted with cold lysis buffer. Following centrifugation, the supernatant was harvested. Subsequently, 20µg protein samples were electrophoresed on 6%~12% SDS-PAGE, and transferred onto nitrocellulose membrane (Millipore, USA). After blocked with 5% nonfat milk in Tris-buffered saline buffer (20mM Tris, 150mM NaCl, pH 7.6, Tween20 0.1%), the membranes were incubated with primary antibodies at 4°C overnight, and reacted with horseradish peroxidase-conjugated secondary antibodies. Blot bands were developed by enhanced chemiluminescence reagents (Amersham, UK). PI3K Class Ⅰ, PI3K Class Ⅲ, E-cadherin, Snail, p-mTOR, mTOR, p-ERK, ERK and β-actin antibodies were purchased from Santa cruz Biotechnology (Santa cruz, CA, USA). Vimentin, N-cadherin, Atg5 and Beclin-1 antibodies were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). U0126, SP600125, p-Akt, Akt, p-JNK, JNK, p-p38 and p38 antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). Antibody against LC3B was purchased from Abcam (Cambridge, UK).
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