Anti-LC3B antibody produced in rabbit

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- Lyse cells in 1 × SDS sample buffer

Upstream tips
- Lyse cells in 1 × SDS sample buffer

Publication protocol

Immunofluorescent staining:
Kidney cryosections at 3 μm thickness were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% TritonX-100 in PBS for 5 min at RT. After blocking with 2% donkey serum for 60 min, the slides were immunostained with primary antibodies against LC3-β (cat: L7543, Sigma Aldrich, St Louis, MO), cleaved caspase3 (cat: 9664, Cell Signaling Technology, Beverly, MA) and F4/80 (cat: 14–4801, eBioscience, San Diego, CA). The slides were then stained with TRITC or FITC-conjugated secondary antibodies. Slides were viewed with a Nikon Eclipse 80i Epifluorescence microscope equipped with a digital camera.

Western blot analysis:
Cultural NRK-52E cells were lysed in 1 × SDS sample buffer. The kidneys were lysed with RIPA solution containing 1% NP40, 0.1%SDS, 100 mg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma, St Louis, MO) on ice. The supernatants were collected after centrifugation at 13,000 × g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay. An equal amount of protein was loaded into 10% or 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-LC3-β (cat: L7543, Sigma Aldrich, St Louis, MO), anti-cleaved caspase3 (cat: 9664, Cell Signaling Technology, Beverly, MA), anti-β-actin (cat: sc-1616, Santa Cruz Biotechnology), anti-p-AMPKα (T172) (cat: 2535, Cell Signaling Technology, Beverly, MA), anti-AMPKα (cat: 5831, Cell Signaling Technology, Beverly, MA), anti-p-S6 (S235/236) (cat: 4857, Cell Signaling Technology, Beverly, MA). Quantification was performed by measuring the intensity of the signals with the aid of National Institutes of Health Image software package.

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