The cells transfected with different siRNAs were harvested following 24 hours of transfection and washed once with cold PBS. Cells were then suspended in an appropriate volume of lysis buffer (20 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA,1 mM EGTA, 1 mM PMSF and 1% Triton X-100) and incubated for 30 minutes at 4 °C. Cells were then centrifuged at the highest speed for 15 minutes and the supernatant was collected for quantification using the BCA protein quantification kit (Pierce, USA). 20 μg of proteins were separated by 4–12%% SDSPAGE and then transferred onto nitrocellulose membranes (Amersham Pharmacia, UK). Rabl3 antibody (Proteintech, USA, 1:1000), LC3 antibody (Cell Signaling, USA, 1:1000), MAPK8 (Cell Signaling, USA, 1:2000), MAPK14 (Proteintech, USA, 1:2000), p-MAPK8/9/10 (Cell Signaling, USA, 1:1000) and p-MAPK11/12/13/14 (Cell Signaling, USA, 1:1000) and Actin Beta Antibody (Sigma, USA, 1:5000) were used.
Lc3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The LC3 antibody is a primary antibody used to detect the microtubule-associated protein 1A/1B-light chain 3 (LC3) in various research applications. LC3 is a widely accepted marker for autophagosomes and is essential for the formation and expansion of these structures during the process of autophagy.
Automatically generated - may contain errors
Lab products found in correlation
Pchk1 s345, by Cell Signaling Technology (2 mentions)
Lamin a c antibody, by Abcam (2 mentions)
Pegfp chk1, by Addgene (2 mentions)
Pgk hl2a, by Addgene (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Abcam (2 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Methanol, by Merck Group (2 mentions)
Normal goat serum (ngs), by Cell Signaling Technology (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Mapk8, by Cell Signaling Technology (1 mentions)
Mapk14, by Proteintech (1 mentions)
Horseradish peroxidase labeled goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Antibody to actin, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Fv10i, by Olympus (1 mentions)
Cleaved caspase 3 antibody, by Proteintech (1 mentions)
30 protocols using lc3 antibody
1
Western Blot Analysis of Autophagy and MAPK Signaling
2
Quantifying Autophagic Protein Expression
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After collecting Tregs from each group (n = 15/group), total protein was extracted, and the protein concentration was measured by the bicinchoninic acid method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for ~1.5 h. Then, proteins were transferred to a nitrocellulose membrane using a semi-dry method for ~50 min. After the transfer, 5% bovine serum albumin was incubated with the membrane at room temperature on a shaker for 30 min and then incubated overnight with LC3 antibody (Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Hangzhou, China). Tris-buffered saline containing Tween 20 was incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (Cell Signaling Technology) for 1 h at room temperature (20–25°C); electrochemiluminescence detection was performed to evaluate the protein signal by exposure to a gel imager. Protein levels were normalized to GAPDH, and changes were determined such that the LC3-II/GAPDH ratio reflected the degree of autophagic protein expression. LC3-II is more hydrophobic than GADPH, and thus LC3-I appeared as the upper band in the electropherogram, while LC3-II was in the lower band. The ratio of LC3-II/GAPDH was calculated based on GAPDH as the internal reference level of expression for statistical analysis.
3
Detailed Immunostaining and Transfection Protocols
4
Quantification of PARP, LC3, and Grp Proteins
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For analysis of poly ADP-ribose polymerase (PARP) cleavage, light chain 3 (LC3), and glucose-regulated protein (Grp) 78 and Grp94 levels, proteins were separated on 8% denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinyl difluoride membrane, incubated with a 1:1,000 dilution of PARP C-2–10 monoclonal antibody or KDEL antibody to detect Grp78 and Grp94 from Enzo Life Sciences (Farmingdale, NY), LC3 antibody from Cell Signaling Technologies (Danvers, MA), and the antibody to actin used for normalization was from Santa Cruz Biotechnology (Dallas, TX). These antibodies were detected with a 1:10,000 dilution of the corresponding horseradish peroxidase-conjugated secondary antibody from Jackson ImmunoResearch Laboratories (West Grove, PA), and Enhanced Chemiluminescence reagents (EMD Millipore, Billerica, MA) and band intensities were recorded using a CCD camera-based imager (MicroChemi 4.2; FroggaBio, Toronto, ON, Canada). Where required, band intensities were corrected for unequal loading by reprobing the blots for actin.
5
Immunofluorescent Analysis of Autophagy Markers
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The cells were seeded on cover slips in a 6-well plate and then were treated with CA, TMZ, or both at the indicated concentrations for 24 h. The cells were fixed with 4% paraformaldehyde for 40 min under room temperature and were then stained with LC3 antibody (1:100, #2775, Cell Signalling Technology, MA, USA). The nuclei were counterstained with 0.1 µg/ml DAPI. Cells were then stained with a secondary antibody (1:1000, #A-11034, Thermo Fisher Scientific, MA, USA). The coverslips were viewed using confocal microscopy (FV10i, Olympus, Tokyo, Japan) with appropriate filters.
6
Detailed Immunostaining and Transfection Protocols
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Sources of chemicals were as previously described15 (link),19 (link),30 (link). The following antibodies were used at the indicated dilutions for immunofluorescence (IF) or immunoblot (IB): antibodies against γH2AX and H2AX (1:500 (IF) and 1:1000 (IB)) were from Cell Signaling; the antibody against the cytosolic tail of L2A (1:100 (IF) and 1:5,000 (IB)) was from Invitrogen, against LAMP1 (1:100 (IF) and 1:1,000 (IB)) was from Iowa University Hybridoma Bank, against hsc70 (13D3, 1:1,000) from Novus Biotechnology, against GAPDH (1,5000) and actin (1:5,000) from Abcam, against GFP (1:100 (IF) and 1:3,000 (IB)) from Roche, against p62 (1:3,000 (IB)) from Biomol international, against pChk1S345, pChk1S317, Chk1, Chk2, Mre11, Nbs1, Rad50, ATM and ATR (1:1,000 (IB), and 1:100 (IF)) from Cell Signaling. LC3 antibody was from cell Signaling, used at 1:2,000 (IB). Lamin A/C antibody was from abcam, used at 1:3,000 (IB). The plasmid for Chk1 (peGFP-Chk1) was from Addgene and the plasmid for human L2A (pGK-hL2A) was generated in our laboratory31 (link). Cells were transfected with cDNA constructs using Lipofectamine™ 2000 reagent (Invitrogen) according to manufacturer’s instructions. Transfection efficiency was monitorized by co-expression of a different fluorescent protein. Mutagenesis was performed using the Quikchange II Site-directed mutagenesis kit (Agilent Technologies).
7
Autophagy Induction and Visualization
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HEK293T cells were transfected with 2 μg of polyI:C or infected with SeV at 200 HA units/mL, and collected after 12 h. Subsequently, Cyto-ID autophagy reagent (Enzo, #ENZ-51031, Farmingdale, NY, USA) staining was performed according to the instruction of the manufacturer. Briefly, the cells were washed twice with 1 × assay buffer and treated with diluted Cyto-ID green stain solution. The cells were incubated for 30 min at 37°C, and then washed and incubated for 20 min with 4% paraformaldehyde. For LC3 staining, the cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% tritonX-100 buffer. Then cells were stained with LC3 antibody (Cell Signaling Technology, #3868) for 2 h at 37°C and stained with anti-rabbit IgG FITC reagent. The cells were then washed three times and analyzed by fluorescence microscopy to observe punctated forms of autophagosomes.
8
Immunohistochemistry of Formalin-Fixed Specimens
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We made 4 μm-thick sections of formalin-fixed paraffin-embedded specimens for IHC. Slides were deparaffinized in xylene and rehydrated with 100% ethanol, 95% ethanol and water serially. We immersed slides into antigen-retrieval buffer (10 mM Tri-sodium citrate dihydrate, 0.05% Tween-20, pH 6.0) boiled via water bath for 30 minutes, and stained the slides using an UltraVision Quanto HRP Detection kit (Thermo Scientific, Rockford, IL, USA) with specific antibodies according to the manufacturer’s instructions. Antibodies of CD45, iNOS were from Abcam, Cleaved caspase-3 antibody was from Proteintech (Rosemont, IL, USA), LC3 antibody was from Cell Signaling Technology, and beclin-1 antibody was from Bioss (Woburn, Massachusetts, USA). CML antibody was from TransGenic (Tokyo, Japan). Microvascular endothelium in the tissue slides was identified by Cd31 staining.
9
Spinetoram Cytotoxicity Evaluation Protocol
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Spinetoram (CAS No: 187166-40-1, purity > 98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin–streptomycin were obtained from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was obtained from Gibco (Norristown, PA, USA). Phosphate buffered saline (PBS) was obtained from Servicebio (Wuhan, China).
The malondialdehyde (MDA) content assay kit (D799762-0100), reduced glutathione (GSH) content assay kit (D799614-0100), catalase (CAT) activity assay kit (D799598-9100), and superoxide dismutase (SOD) activity assay kit (D799594-0100) were bought from Sangon Biotech (Beijing, China). The LC3 antibody, Beclin1 antibody, p62 antibody, p-mTOR antibody, p-AMPK antibody, GAPDH antibody, and γH2AX antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). The Alexa fluor 488-conjugated antibody were purchased from Sangon Biotech (Shanghai, China).
The malondialdehyde (MDA) content assay kit (D799762-0100), reduced glutathione (GSH) content assay kit (D799614-0100), catalase (CAT) activity assay kit (D799598-9100), and superoxide dismutase (SOD) activity assay kit (D799594-0100) were bought from Sangon Biotech (Beijing, China). The LC3 antibody, Beclin1 antibody, p62 antibody, p-mTOR antibody, p-AMPK antibody, GAPDH antibody, and γH2AX antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). The Alexa fluor 488-conjugated antibody were purchased from Sangon Biotech (Shanghai, China).
10
LC3 Immunofluorescence Staining Protocol
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Cells on coverslips were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin for 1 hour, followed by incubation with LC3 antibody (#4108, 1:500; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing with phosphate-buffered saline, cells were stained with FITC-labeled goat anti-rabbit IgG (ab6717, 1:1,000; Abcam, Cambridge, MA, USA) at 37°C for 1 hour. Cells were observed utilizing a confocal microscopy (Olympus, Tokyo, Japan).
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