LC3A (D50G8) XP® Rabbit mAb

Autophagy assay cell type - Human primary MSCs

Experiment
Autophagy assay cell type - Human primary MSCs
Product
LC3A (D50G8) XP® Rabbit mAb from Cell Signaling Technology
Manufacturer
Cell Signaling Technology

Protocol tips

Protocol tips
For WB, dilutr primary Ab at 1:1000 and for Immunofluorescence at 1:50 and incubate overnight at 4 °C

Publication protocol

Protein extraction and western blotting analysis
Cell extracts were prepared with M-PER (78501, Pierce, Rockford, IL) plus protease inhibitor cocktail (78442, Pierce) and protein concentrations were determined using the BCA assay (23225, Pierce). Protein lysates (30 μg) were separated on SDS polyacrylamide gels, transferred onto PVDF membranes, blocked with 5% blotting grade milk (1706404, Bio-Rad, Hercules, CA) in TBST (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 1% Tween 20). The membranes were incubated with the primary antibodies overnight at 4 °C. After extensive washing, the membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr. Immunoreactive proteins were then developed using an enhanced chemiluminescence detection system (PerkinElmer, Boston, MA). Membranes were exposed to X-ray film (Amersham Pharmacia Biotech, Piscataway, NJ) for visualization. Primary antibodies include anti-phospho-mTOR (Ser2448) (1:1000, BS4706, Bioworld, St Louis, MN), anti-cleaved caspase-3 (1:1000, E83-77, Epitomics), anti-fibronectin (1:5000, SC-8422, Santa Cruz, San Diego, CA), anti-phospho-ULK1 (ser757) (1:1000, 6888, Cell Signaling, Danvers, MA), anti-phospho-S6K (Thr389) (1:1000, 9234, Cell Signaling), anti-cleaved caspase-9 (1:1000, 9502, Cell Signaling), anti-phospho-S6 (Ser235/236) (1:1000, 2211, Cell Signaling), anti-LC3A (1:1000, 4599, Cell Signaling), and phospho-4E-BP1 (1:1000, 2855, Cell Signaling). An anti-β actin antibody (1:5000, NB600-501, Novus, Littleton, CO) was used as loading control.

Immunofluorescence
For immunofluorescence staining, specimens were fixed in PBS with 4% formaldehyde, washed twice with PBS, and immersed in 0.1% Triton X-100 overnight at 4 °C, followed by washing 3 times with PBS. The following primary antibodies were incubated overnight at 4 °C: anti-phospho-mTOR (Ser2448) (1:200, Bioworld), anti-cleaved caspase-3 (1:200, Epitomics), anti-fibronectin (1:200, Santa Cruz) anti-phospho-ULK1 (ser757) (1:1000, Cell Signaling), anti-phospho-S6 (Ser235/236) (1:200, Cell Signaling) or anti-LC3A (1:50, Cell Signaling). After incubation with primary antibodies, cells were washed with PBS and then incubated with fluorescence-conjugated secondary antibodies (1:5000, FITC, Jackson ImmunoResearch, Westgrove, PA) for 2 hr at room temperature. After nuclear staining with 40,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich), spheres were then placed in a Lab-Tek®II chamber (Nunc, Naperville, IL) and analyzed with a confocal fluorescent microscope (Olympus FV10i). Negative controls without utilizing primary antibodies were also prepared to rule out nonspecific labeling.

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Papers

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Paper title
Involvement of mTOR-autophagy in the selection of primitive mesenchymal stem cells in chitosan film 3-dimensional culture
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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A (D50G8) XP® Rabbit mAb below.

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