Anti-p62 (SQSTM1) (Human) pAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For WB, dilute Ab at 1:100 and for ICC at 1:500

Protocol tips
For WB, dilute Ab at 1:100 and for ICC at 1:500

Publication protocol

Immunoblotting assays
Cell lysates were prepared with RIPA buffer with a protease and phosphatase inhibitor cocktail (Thermo Scientific). Twenty to thirty micrograms of total protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Specific proteins were detected using specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies, together with enhanced chemiluminescence developing agent (Millipore). Images were digitally acquired with a Kodak Image Station 4000 (Carestream Health Inc., Rochester, NY, USA) or ImageQuant LAS 4000mini (GE, Uppsala, Sweden), and the companion software. The density of LC3-II was first normalized to the loading control, tubulin. Then, the ratio of LC3-II/tubulin is converted to numbers relative to that of the control treatment.

Immunofluorescence staining
Cells were grown on 3 cm Petri dish and were fixed in 4% paraformaldehyde for 15 min. Cells were washed two times in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 for 15 min, followed by another wash in PBS and block in PBS containing 5% bovine serum albumin (BSA) for 1 h. Primary and secondary antibodies in PBS containing 5% BSA were applied overnight at 4 °C and 1 h at room temperature, respectively. Nuclei were costained with 1 μg/ml Hoechst 33342. Fluorescence images were taken using a Nikon Eclipse TE200 epifluorescence microscope with NIS-Elements AR3.2 software or EVOS FL Auto (Life Technologies, Bothell, WA, USA). For manual quantification of the puncta formation at least three optical fields with at least 50 cells per experimental condition were analyzed. Data from repeated experiments were subjected to statistical analysis.

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Manufacturer protocol

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