SQSTM1/p62 Antibody

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

Western blotting
Western blot analysis was performed according to our previous protocol [41]. Briefly, both vascular tissue samples obtained from ApoE-/- mice and THP-1-derived macrophages treated with PBS or ox-LDL were harvested, and added to Cell Lysis Buffer for Western and IP with both protease and phosphatase inhibitors following the manufacturers’ instructions. The homogenate was centrifuged at 14,000 x g for 15 min at 4°C. The protein concentration was measured using a BCA protein assay kit according to the manufacturer's protocol. Same amounts of total protein were loaded in each lane of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% gels and then were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked for 4 hrs with 5 % (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) at room temperature and incubated overnight with the primary antibodies at 4°C. The membranes were washed with TBST, incubated with appropriate HRP-conjugated secondary anti-mouse or -rabbit antibodies for 2 hrs at room temperature, and rewashed with TBST for 30 min (three times for 10 min each). An enhanced chemiluminescence solution was used to develop the blots for 5 min. Target proteins were detected using an enhanced chemiluminescence detection system (MiniChemi, Beijing Sage Creation Science Co., Ltd., Beijing, China). Pre-determined molecular weight standards were used as markers. Relative protein expression levels were semiquantitatively performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunoprecipitation
For immunoprecipitation analysis of different treated cells, protein lysates (2 mg) were prepared from THP-1 cells and mixed with the anti-acetyl lysine antibody (No.: A2391, 5 μL) on an ice cold shaker overnight, which was followed by the addition of adequately resuspended protein A/G magnetic beads (60 μL, Biotool) for another 2 hrs at 4°C. Immune complexes were washed three times with wash buffer and then 30 μL of elution buffer was added. After boiling in 4 x sample buffer, samples were subjected to denaturing SDS-PAGE analysis of Atg5.

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Manufacturer protocol

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