SQSTM1/p62 (D5E2) Rabbit mAb

Protocol tips

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	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Publication protocol

Cells were lysed in ice-cold RIPA lysis buffer (POO13C; Beyotime, Shanghai, China). The protein concentration was determined by BCA method. The total protein samples (40 µg) were separated on 12% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with Tris-buffered saline and Tween-20 containing 5% non-fat dry skimmed-milk for 1 h at room temperature, membranes were incubated overnight with the previously mentioned primary antibodies (p62/SQSTM1, 1:1,000 dilution; LC3B, 1:1,000 dilution; and β-actin, 1:1,000 dilution) at 4°C. The appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG, 1:5,000 dilution; goat anti-rabbbit IgG, 1:5,000 dilution) were applied for 1 h at room temperature, and immunoreactivity was subsequently visualized using a WesternBright enhanced chemiluminescent kit (Advansta, Inc., Menlo Park, CA, USA). Furthermore, densitometric analyses were performed using an imaging analyzer (Tanon 5200; Tanon Science and Technology Co., Ltd., Shanghai, China) and Tanon Gis software (Gel Image System Ver. 4.00; Tanon Science and Technology Co., Ltd.). Experiments were performed in triplicate.

Based on the method described above, firstly, the conversion of LC3-I to LC3-II was determined by western blotting in order to evaluate the activity of autophagic initiation. Subsequently, the activity of autophagic flux was assessed by using chloroquine (CQ), a specific lysosomal inhibitor that interferes with vesicular acidification. A total of 2×105 cells/well were grown in 6-well plates with 2 ml serum-free RPMI-1640 and transfected with 50 nM con siRNA or p62 siRNA for 48 h using a HiPerfect Transfection reagent. Western blotting was subsequently performed with the anti-LC3B antibody following exposure to 20 µg/ml oxLDL for the indicated time, with or without CQ (30 µM) for the last 2 h.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for SQSTM1/p62 (D5E2) Rabbit mAb below.

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