LC3B antibody

Protocol tips

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	- Dilute Ab at 1:1000 and incubate at room temperature for 1h

Protocol tips
- Dilute Ab at 1:1000 and incubate at room temperature for 1h

Publication protocol

IEC-18 cells were seeded into 6-well plates and subjected to the respective treatments. After a 6-h incubation, the media were removed, and the cells were washed thrice with ice-cold PBS (pH 7.4). Adherent cells were collected with a cell scraper and lysed in radioimmunoprecipitation assay buffer containing 1% phosphatase inhibitors (Applygen Technologies Inc., Beijing, China) on ice for 30 min with intermittent vortexing (5-7 times, 30 s each, at 5-min intervals). Then, the cell lysates were centrifuged at 12, 000 x g for 15 min at 4 °C, and the supernatants were collected on ice. The protein concentration was measured using a Pierce BCA protein assay kit (Thermo Scientific), and the samples were stored at -80 °C until needed. Protein samples (30 µg/well) from each treatment group were separated by 12% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (0.45 um). The membranes were blocked with 10% (w/v) skim milk in 1× Tris-buffered saline and Tween-20 (pH 7.4) at room temperature for 1 h and then probed separately with LC3B (1:1000; Cell Signaling Technology, Danvers, MA, USA) and P62 (1:1000; Cell Signaling Technology) antibodies at 4 °C overnight. The next day, the membranes were washed thrice for 10 min per wash and incubated with a goat anti-rabbit IgG secondary antibody (HRP) (1:5000; Jackson ImmunoResearch Inc.) at room temperature for 1 h. Then, the membranes were washed thrice as described above, and the antibody signals were detected by autoradiography using a PierceTM ECL Western Blotting Substrate (Thermo Scientific). Detection of LC3-II was used to evaluate the level of activated LC3. Densitometric analysis of the blots was conducted using the FluorChem FC3 system (ProteinSimple, California, USA).

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