Cells and tissues were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Sigma-Aldrich). After centrifugation at 13,000 g for 20 min, the supernatant was collected and quantified using a BCA protein assay kit (Thermo Fisher Scientific). Total protein (50 µg) was subjected to electrophoresis on a 10% SDS polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Molsheim, France). After being blocked in 5% non-fat powdered milk, the membranes were probed with primary antibodies at the indicated dilution, followed by the incubation with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Abcam, Cambridge, UK). Protein bands were detected using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and analyzed by GeneTools software (Syngene, Cambridge, UK). The following antibodies were used: anti-Beclin1 (Abcam; dilution 1:1,000), anti-light chain 3 I (anti-LC3 I, Cell Signaling Technology, Danvers, MA, USA; dilution 1:1,000), anti-LC3 II (Cell Signaling Technology; dilution 1:1,000), anti-Bcl-2 (Abcam; dilution 1:1,000), anti-Bax (Abcam; dilution 1:1,000), and anti-VASP (Abcam; dilution 1:3,000). Expression of glyceraldehyde-3-phosphate dehydrogenase was used as a loading control.
Anti lc3 2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-LC3-II is a primary antibody that specifically detects the lipidated form of the autophagy marker protein LC3 (LC3-II). It can be used to monitor autophagy levels in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti p62, by Cell Signaling Technology (5 mentions)
Anti lc3 1, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti lamp2, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti beclin1, by Abcam (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse or anti rabbit igg secondary antibodies, by Abcam (1 mentions)
Anti vasp, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Genetools software, by Syngene (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti mtf 1, by Abcam (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Bicinchoninic assay kit, by Thermo Fisher Scientific (1 mentions)
24 protocols using anti lc3 2
1
Quantitative Analysis of Autophagy and Apoptosis Markers
2
Western Blot Analysis of Metallothionein
3
Cartilage Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein was extracted from 10 mg articular cartilage using a protein isolation kit (ReadyPrep; GE Healthcare Life Sciences). The protein concentration was determined using a bicinchoninic assay kit (Thermo Fisher Scientific, Inc.). The protein (20 μg) was separated on 12% SDS PAGE gel and transferred to the nitrocellulose membrane. At room temperature, the membrane was sealed in 5% skimmed milk for 2 hours and incubated with the following primary antibodies at 4°C overnight: Anti-LC3 I, Anti-LC3 II, anti-MMP13, anti-aggrecan, anti-collagen II, anti-TGFβR1, anti-β-actin (Cell Signaling Technology, Inc., Danvers, USA). The next day, the nitrocellulose membranes were washed three times and incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000, cat. no. A16104SAMPLE; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The protein bands were detected by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) scanned by ChemiDoc XRS (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein expression was normalized to β-actin and densitometric analysis was performed by ImageJ Software version 7.0 (National Institutes of Health, Bethesda, MD, USA).
4
Comprehensive Immunomodulatory Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chloroquine (C6628) was purchased from Sigma Aldrich. E64 (S7379) was purchased from Selleckchem. Pepstatin (M183) was purchased from AMRESCO. Annexin-V-FITC was synthesized by our laboratory. Propidium iodide (PI) was purchased from Sunshine Biotechnology. Lipofectamine 2000 was purchased from Invitrogen. The antibody used were as follows. Anti-LC3-II (2775) was purchased from Cell Signaling Technology. Gapdh (D3015) was purchased from Santa Cruz Biotechnology. Anti-CD4-PE (557308) and Anti-Gr-FITC (551461) were purchased from BD. Anti-CD8-APC (E07056-1633) and anti-F4/80-FITC (E00611-1636) were purchased from eBioscience.
5
Immunoblotting Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extracts for immunoblotting were acquired from N2a cells homogenized in RIPA buffer (radioimmunoprecipitation assay buffer, Thermo Fisher Scientific, Catalog Numbers 89900). Briefly, proteins were separated by electrophoresis and subsequently transferred to the PVDF membrane by electroblotting using standard protocols. The primary antibodies used were anti-LC3II (Cell Signaling Technology, catalog #2775s), anti-COX IV (Proteintech, catalog no. 11242–1-AP), anti-TOM40 (Proteintech, catalog no. 18409–1-AP), anti-MnSOD (Proteintech, catalog no. 24127–1-AP), anti-CLS (Proteintech, catalog no.14845–1-AP), anti-PLS3 (Proteintech, catalog no.28028–1-AP), anti-NDPK-D (Bioss, bs-11902R), anti-PINK1 (Novus Biologicals, catalog no. BC100–494), anti-Parkin (Abcam, catalog no. ab77924), anti-Drp1 (Cell Signaling Technology, catalog #8570), anti-Lamp2 (Cell Signaling Technology, catalog #49067), PGC1a monoclonal antibody (Proteintech, catalog no. 66369–1-Ig), recombinant anti-mtTFA antibody (Abcam, ab252432), GAPDH monoclonal antibody (Proteintech, catalog no. 60004–1-Ig), and anti-Tubulin (Proteintech, catalog no. 11224–1-AP).
6
Cardiomyocyte Apoptosis and Autophagy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The apoptosis and autophagy of cardiomyocytes were evaluated by Immunofluorescence staining of frozen sections with anti-active caspase-3 (rabbit polyclonal, 1:100; Abcam, Cambridge, MA, USA) or anti-LC3-II (1:200; Cell Signal Technology, Danvers, MA, USA) according to the manufacturer's direction. The sections derived from three to four slides of each group were examined and calculated.
7
Immunofluorescence Assay of Hsp90 Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunofluorescence assays of Hsp90 were performed using the aforementioned treatments. Then samples were fixed (4% paraformaldehyde), permeabilized (20–30 min 0.05%Triton-X in PBS 1×) and incubated overnight at 4°C with different primary antibodies anti-Alix (1 : 100, sc53540), anti-Flotillin-1 (1 : 100, sc74566), antiCD9 (1 : 100, donation from Sanchez-Madrid), anti-LC3-II (Cell Signaling #2775) and anti-caspase-2 (ab182657). The samples were washed with 0.05% Tween 20 in PBS, incubated for 30 min in the specific secondary antibody conjugated with Alexa-568 (ThermoFisher), washed in PBS and mounted with the Vectashield antifade mounting medium (Vector).
Confocal images were obtained in the microscopy facility (IDIVAL, Santander, Spain) with a confocal microscope NIKON A1R spectral (405 nm, 488 nm, 514 nm, 561 nm, 638 nm) and Fiji52 (link) was used for data visualization.
Confocal images were obtained in the microscopy facility (IDIVAL, Santander, Spain) with a confocal microscope NIKON A1R spectral (405 nm, 488 nm, 514 nm, 561 nm, 638 nm) and Fiji52 (link) was used for data visualization.
8
Mitochondrial Dynamics and Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemical reagents used in this study were Mito Green Tracker (Beyotime, C1048). Primary antibodies used in this study include the following: rabbit polyclonal anti-Drp1 (Proteintech, #12957-1-AP, 1: 500 dilution); rabbit polyclonal anti-Drp1(Ser616) (Cell Signaling, #3455, 1:1000 dilution); rabbit polyclonal anti-PINK1 (Cell Signaling, #6946, 1:1000 dilution); rabbit monoclonal anti-Parkin (abcam, ab15954, 1:1000 dilution); rabbit polyclonal anti-Mfn1 (Proteintech, #13798-1-AP, 1: 500 dilution); rabbit monoclonal anti-LC3 II (Cell Signaling, #3868, 1:1000 dilution); rabbit anti-cleaved Caspase3 (Cell Signaling, #9664, 1:500 dilution); rabbit anti-cleaved PARP (Cell Signaling, #9509, 1:500 dilution); mouse monoclonal anti-GAPDH (Proteintech, #60004-1-lg, 1:1000 dilution); rabbit polyclonal anti-Tom20 (Proteintech, #11802-1-AP, 1:1000 dilution); mouse polyclonal anti-PRRSV Nsp2 has been described previously [39 (link)]. The secondary antibodies used for immunoblot analysis were HRP-conjugated anti-mouse IgG (BOSTER), HRP-conjugated anti-rabbit IgG (Cell Signaling). The secondary antibodies used for immunofluorescence was Cy3-conjugated anti-mouse IgG (BOSTER), FITC-conjugated anti-mouse IgG (BOSTER).
9
Visualizing Autophagy Regulation by USP24
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells seeded in 6-well plates with cover slips inside were infected with scramble and shUSP24-knockdown lentivirus (m.o.i. = 5) for 48 h. The cells in coverslips were fixed with 4% paraformaldehyde at 4 °C for 15 min. After fixation, coverslips were washed with PBS, and incubated with 0.2% Triton X-100 in PBS for 5 min at room temperature, then were blocked with 1% Bovine serum albumin (BSA) for 1 h, and stained with anti-GFP (1:200) (SC-9996, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LC3-II (1:200) (#3868, Cell Signaling technology, Danvers, MA, USA) for 16 h at 4 °C. After washing with PBS, cells were stained with Alexa Fluor® 488 or 568 (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature and mounted with 90% glycerol containing DAPI (Invitrogen, Carlsbad, CA, USA). The number of stained cells were examined by fluorescence microscopy (Olympus, Tokyo, Japan.). ImageJ (Bethesda, Maryland, USA.) was used to perform the statistical analysis.
10
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell cultures were washed twice with cold PBS and homogenized in lysis buffer (4% SDS, 2.1 mM EDTA, and 50 mM Tris). Aliquots were taken for protein determination (Peterson, 1983 (link)), and β-mercaptoethanol was added to a final concentration of 5%. Thirty micrograms of protein was separated on 12% SDS–PAGE (Bio-Rad, Hercules, CA) and electrotransferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% M-TTBS (5% milk in Tween-20 in Tris-buffered saline [TTBS]) and further incubated with anti-P2×7 antibody (1:500; Santa Cruz Biotechnology), anti-Bcl2 (1:1000), anti-pAMPK (1:1000), anti–cleaved PARP (1:1000), anti-p62 (1:1000), anti-p53 (1:1000), and anti-LC3II (1:1000; Cell Signaling, Danvers, MA), diluted in TTBS, at room temperature. The membranes were then incubated with horseradish peroxidase–conjugated secondary antibody (1:2000) for 2 h at room temperature, and chemiluminescence was detected using x-ray films (X-Omat; Kodak, Rochester, NY). The films were scanned, and the percentage of band intensity was analyzed using ImageJ (National Institutes of Health, Bethesda, MD).
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!