SQSTM1/p62 (D5L7G) Mouse mAb #88588

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For WB , dilute primary Ab at 1:1000 and for immunoflourence at 1:800. Incubate at 4°C overnight for WB

Protocol tips
For WB , dilute primary Ab at 1:1000 and for immunoflourence at 1:800. Incubate at 4°C overnight for WB

Publication protocol

Cells lysate preparation and western blot analysis
After 10 μM MTH-3 treatments at indicated intervals of time, MDA-MB-231 cells were harvested, washed and suspended in the PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Gyeonggi-do, Korea). Protein concentrations were estimated using the Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The samples were resolved with SDS-PAGE and transferred to a polyvinylidene difluo-ride membrane (PVDF) (EMD Millipore, Billerica, MA, USA). Each membrane was blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 h followed by individual incubation with specific primary antibodies [cyclin B1 (cat. no. 4138, 1:1,000), CDK1/Cdc2 (cat. no. 9116, 1:1,000), DR3 (cat. no. 4758, 1:1,000), DR5 (cat. no. 8074, 1:1,000), FADD (cat. no. 2782, 1:1,000), Bcl-2 (cat. no. 4223, 1:1,000), Bcl-xL (cat. no. 2764, 1:1,000), Ero1 (cat. no. 3264, 1:1,000), PDI (cat. no. 3501, 1:1,000), PERK (cat. no. 5683, 1:1,000), calnexin (cat. no. 2679, 1:1,000), IRE1α (cat. no. 3294, 1:1,000), CHOP (cat. no. 2895, 1:1,000), Bip (cat. no. 3177, 1:1,000), Atg5 (cat. no. 12994, 1:1,000), Atg7 (cat. no. 8558, 1:1,000), Atg12 (cat. no. 4180, 1:1,000), Beclin-1 (cat. no. 3495, 1:1,000), p62 (cat. no. 88588, 1:1,000), LC3A/B (cat. no. 12741, 1:1,000) and β-actin (cat. no. 3700, 1:5,000) (Cell Signaling Technology, Danvers, MA, USA)] at 4°C overnight. Each membrane was then incubated with anti-rabbit IgG (cat. no. 7074, 1:10,000) or anti-mouse IgG (cat. no. 7076, 1:10,000) horseradish peroxidase (HRP)-linked antibodies (Cell Signaling Technology) at room temperature for 1 h. The signal was detected with the Immobilon Western Chemiluminescent HRP substrate (EMD Millipore) and visualized using the LAS 4000 imaging system (Fuji, Tokyo, Japan) as previously described (36–38). The quantitative densitometric analysis of immunoreactive band was employed by ImageJ bundled with 64-bit Java 1.6.0_24 program for Windows from the National Institutes of Health (NIH; Bethesda, MD, USA).

Immunofluorescence staining
MDA-MB-231 cells (2×106 cells/dish) were grown on sterile coverslips placed in a 10-cm dish. After 10 μM MTH-3 treatment, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS). After blocking with 2% bovine serum albumin (BSA) in PBS, LC3B and p62 were detected using anti-LC3B and anti-p62 antibody followed by reaction with FITC- or PE-conjugated secondary antibody (BD Biosciences). Coverslips were mounted on glass slides with ProLong Gold Antifade reagents (Thermo Fisher Scientific) containing DAPI, and fluorescent image was taken on a Leica Microsystems TCS SP2 Confocal Spectral microscope as detailed by Lu et al (39).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for SQSTM1/p62 (D5L7G) Mouse mAb #88588 below.

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