LC3A/B (D3U4C) XP® Rabbit mAb #12741

Protocol tips

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	1:1000 dilution of primary antibody

Protocol tips
1:1000 dilution of primary antibody

Publication protocol

Cells were incubated in the presence or absence of VP (1.25 μg/mL or 7.5 μg/mL) for 0 h and 6 h under four different conditions: (i) light during both treatment and lysis- figure S, (ii) darkness during treatment but light during lysis (Fig. 1A,B,C), (iii) light during treatment but darkness during lysis (Fig. 5) and (iv) darkness during both treatment and lysis (Fig. 1A,B,C). Darkness conditions were strictly applied in a dark room without any presence of ambient or lamp/source light. Lysates from untreated cells were also treated with the 7.5 μg/ml of VP in light and dark. Total protein concentration was determined by DC Protein Assay (Bio-Rad, Philadelphia, PA, US). Equal amounts of total protein (for cell lysates) were loaded on a NUPAGE 4–12% Bis-Tris polyacrylamide gel (Life Technologies, Grand Island, NJ) and subjected to electrophoresis at 150 V for 1 h and 30 min. Electrophoresis was performed in darkness for the dark conditions in lysis and the complete dark condition. The proteins were transferred to a PVDF membrane (Millipore, Billerica, MA) at 100 V for 1 h, cassettes were filled with ice in order to avoid gel overheating, and the membranes were subsequently blocked with non-fat dry milk for 1 h and incubated overnight with primary antibodies against p-62, Diap1,Rock1, LC3A/B at 1:1000, methionine sulfoxide 1:200; and β-actin at 1:2000 (Cell Signaling Technology). The membrane was then washed and incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology) at 1:10,000. Membranes were developed with enhanced chemiluminescence (ECL Select) (GE Healthcare, Wauwatosa). All experiments were performed in three biological independent replicates unless otherwise noted.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A/B (D3U4C) XP® Rabbit mAb #12741 below.

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