Proteins from AMs in each group were extracted and BCA kit (Pierce, Rockford, IL, USA) was used to identify the protein concentration. After adding with lading buffer, the proteins were boiled at 95°C for 10 min. Then, 30 µg of solution was added in each well in the plates, and the proteins were separated by 10% polyacrylamide gel (Beijing Liuyi Biotechnology Co., Ltd., Beijing, China) and transferred to polyvinylidene difluoride (PVDF) membrane followed by blocking with 5% bovine serum albumin at ambient temperature for 1 h. Primary antibodies GRP78 (1:1000, Abcam, Cambridge, MA, USA), PERK, LC3I, LC3II, Beclin1, Bcl-2 and caspase-3 (1: 1000, Cell Signaling Technology, Inc., Beverly, MA, USA) and primary antibody β-actin (loading control, 1:3000, BD Pharmingen, San Jose, CA, USA) were added for overnight at 4°C. The membrane was then washed three times with PBS for 5 min and incubated with secondary antibodies at ambient temperature. Chemiluminescence reagents were used for color development. Gel Doc EZ imager (Bio-rad, California, USA) was used for color observation. Image J software (National Institutes of Health, Maryland, USA) was used to analyze the gray value of target band. Experiments were conducted for three times to obtain the average value.
Lc3 1
Manufactured by Cell Signaling Technology
Sourced in United States
LC3-I is a protein involved in autophagy, a cellular process that degrades and recycles damaged or unnecessary cellular components. It is a commonly used marker for monitoring autophagy.
Automatically generated - may contain errors
Lab products found in correlation
Lc3 2, by Cell Signaling Technology (16 mentions)
Beclin 1, by Cell Signaling Technology (8 mentions)
Bcl 2, by Cell Signaling Technology (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (4 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Cytochrome c, by Cell Signaling Technology (2 mentions)
Cell counting kit 8 cck 8, by Thermo Fisher Scientific (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (2 mentions)
Annix 5 pi kit, by Thermo Fisher Scientific (2 mentions)
Jc 1 kit, by Thermo Fisher Scientific (2 mentions)
Ros kit, by Thermo Fisher Scientific (2 mentions)
Apoptotic protease activating factor 1 apaf 1, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by Huabio (2 mentions)
Cleaved caspase 9, by Cell Signaling Technology (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
β actin, by BD (1 mentions)
16 protocols using lc3 1
1
Protein Expression Analysis in AMs
2
Endoplasmic Reticulum Stress Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies to GRP78, IRE1α, PERK, eIF2α, ATF4, Bcl-2, Bax, LC3II, LC3I, p62, and phospho-specific antibodies to p-IRE1α/p-PERK p-eIF2α and p-JNK were from Cell Signaling Technology (Beverly, MA, USA). Anticaspase 9, CHOP, BDNF, SYN, and AChR antibodies were obtained from Abcam Inc. (Cambridge, MA, USA). Anti-Beclin-1 antibody was purchased from Novus Biologicals (Littleton, San Diego, USA). Anti-FATP1 antibody was obtained from Santa Cruz Biotechnology (Dallas, Texas, CA, USA). Palmitic acid (PA) and 4-phenylbutyrate (4-PBA) were from Sigma-Aldrich Corp. (St. Louis, MO, USA). 3-Methyladenine (3-MA) was purchased from MedChemExpress (HY-19312, New Jersey, USA).
3
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The collected tissues were lysed in a lysis buffer (150 mM Tris, 50 mM DTT, 8 M urea, 15% sucrose, 2% sodium dodecyl sulfate, 0.01% bromophenol blue, 2 mM EDTA, and a 1% cocktail of protease and phosphatase inhibitors) and sonicated for 3 min. In the next step, the lysate was centrifuged at 12 000 g for 15 min, and the supernatant was collected and subjected to boiling. Subsequently, an equal amount of proteins from the samples was separated by SDS-PAGE and transferred onto a PMSF membrane (Amersham Biosciences, Piscataway, NJ, USA), which was then blocked with TBST (50 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween-20) containing 5% skim milk. In the next step, the membrane was incubated at 4°C overnight with diluted primary antibodies (1: 5000) against Nrf2, Beclin-1, LC3-I, LC3-II, Bax, Bcl-2, ac-SOD and Cleaved Caspase-3 (Cell Signaling Technologies, Danvers, MA, USA). The membrane was then rinsed and incubated with secondary antibodies (1: 5000 dilution, Sigma-Aldrich Co., LLC, USA) at room temperature for 1.5 h. Finally, the density of protein bands on the membrane was measured using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA) and quantified with Quantity One software (West Berkeley, CA, USA). All experiments were carried out in triplicate.
4
Western Blot Analysis of Cellular Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and the cell lysates were prepared using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific). After determination of protein concentration with a BCA assay kit, equal amount of protein samples were separated by 10% SDS-PAGE. Then the proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% non-fat milk in TBST for 1 hour at room temperature. Then the membranes were incubated at 4°C overnight with the primary antibodies against the following proteins: E-cadherin, N-cadherin, vimentin, cleaved caspase-3, p53, Bax, Bcl-2, LC3I, LC3II, p-ERK, ERK, Akt, and p-Akt (diluted in 1:1,000; Cell Signaling Technology, Boston, MA, USA); ABCB1, ABCG2, phosphatidylinositol 3-kinase (PI3K), p-PI3K, mammalian target of rapamycin (mTOR), p-mTOR, and GAPDH (diluted in 1:800; Abcam, Cambridge, MA, USA). Then, the membranes were probed with secondary antibody (diluted in 1:3,000; Abcam) conjugated to horseradish peroxidase. The bands were visualized with a Plus-ECL kit (PerkinElmer, Waltham, MA, USA). The experiment was performed in triplicate.
5
Assessing Apoptosis and Autophagy in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM/F12 high glucose was provided by Hyclone (Utah, USA). Fatal bovine serum(FBS), trypsin, penicillin-streptomycin, Annix-V/PI kit, Cell Counting Kit-8(CCK-8), JC-1 kit and ROS kit were obtained from Gibco (New York, USA). Transwell chambers with membranes at a pore size of 8 μm were bought from Corning Life Science (New York, USA). Hoechst 33258 was purchased from Sigma-Aldrich (St Louis, MO, USA). PI was purchased from MultiSciences (Zhejiang Province, China). Primary antibodies against p53, p27, cleaved caspase-9, cleaved caspase-3, Bcl-2, Bax, cyclinD1, cyclin-dependent kinase 2 (CDK2), cytochrome c, apoptotic protease activating factor 1 (Apaf-1), LC3-I, LC3-II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were provided by Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were provided by Huabio (Hangzhou, Zhejiang, China). Pifithrin-α (PFT-α) was provided by Selleck Chemicals (Shanghai, China). PNS was provided by Kunming Pharmaceutical Company (Kunming, China).
6
Mesalazine Enema Efficacy in Colitis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mesalazine enemas (Salofalk, 60 g: 4 g, manufactured by Vifor AG Zweigniederlassung Medichemie Ettingen, Switzerland; import drug license number: H20150127) were purchased from Shenzhen Kangzhe Pharmaceutical Co. Dextran sodium sulfate (DSS), NF-κB p65, Beclin 1, p-NF-κB p65, LC3II, LC3I, and β-actin antibodies were purchased from Cell Signaling Technology (CST). Antibodies against 3-Methyladenine (3-MA), rapamycin (RAPA), and pyrrolidine dithiocarbamate (PDTC) were purchased from Malone Pharma Consulting.
7
Western Blot Analysis of Lung Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung homogenates were prepared and the total protein content in the supernatant of each sample was measured by BCA protein assay (Life Technologies) according to the manufacturer’s instructions. The protein samples were then separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Beyotime, China). The membranes were blocked with 5% non-fat milk and incubated with primary antibodies against SIRT1 (1:5000), LC3-I (1:5000), LC3-II (1:5000), Beclin1 (1:5000), CHOP (1:2000), GRP78 (1:2000), and β-actin (1:10,000) (all Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Following three washes with 0.2% TBS-T, the membranes were incubated with secondary antibodies (horseradish peroxidase-goat anti-rat, 1:10,000; Cell Signaling Technology) for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence system (Pierce Biotechnology, Rockford, MN, USA). β-actin was used as an internal control.
8
Autophagy Marker Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of the autophagy markers LC3-I and LC3-II (Cell Signaling Technology, Inc., cat. no. 2775), SQSTM1/p62 (Cell Signaling Technology, Inc., cat. no. 8025), ATG5 (Cell Signaling Technology, Inc., cat. no. 2630) and Beclin-1 (Cell Signaling Technology, Inc., cat. no. 3738) was assessed using total cell extract. Proteins (30 µg) were resolved on 10% SDS-PAGE, blotted on nitrocellulose membranes and then incubated with the appropriate primary antibodies and secondary antibody, Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, cat. no. 111-035-003).
9
Isolation and Characterization of Ginsenoside F2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ginsenoside F2 was isolated previously from leaves of Panax ginseng by a series of chromatographic procedures [9 (link)]. Ginsenoside F2 has a molecular mass of 784 Da and was isolated with 98% purity. Primary antibodies of PRR5, CISD2, Bcl-2L, NLRX1, RPS15, RPL26, p53, PUMA, Beclin-1, UVRAG, AMBRA-1, mTOR, LC3-II, LC3-I, and β-actin together with all secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The Atg5, Atg7, and Atg10 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
10
Molecular Pathways in Cell Death
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IRI, 3-MA, CQ, N-acetyl-l -cysteine (NAC; a standard antioxidant), SB203580 (a p38 inhibitor), and SP600125 (a JNK inhibitor) were purchased from Sigma (St. Louis, MO, USA), whereas the RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against cleaved PARP, cleaved caspase 3, LC3-I, LC3-II, and β-actin were acquired from Cell Signaling Technology (Danvers, MA, USA), and the corresponding secondary antibodies from Jackson Immunoresearch (West Grove, PA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!