Anti-p62/SQSTM1 antibody produced in rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Seed 1×10^6 cells per 100-mm culture dish.	- Primary Ab dilution-1:1000

Upstream tips
- Seed 1×10^6 cells per 100-mm culture dish.
Protocol tips
- Primary Ab dilution-1:1000

Publication protocol

Cells were plated at a density of 1×106 cells per 100-mm culture dish and treated with curcumin or 3MA as previously described. Cells were washed twice with PBS and lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1.0 mM Na3VO4, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin and 4 µg/ml leupeptin, pH 7.5). Lysates were centrifuged and the supernatants diluted in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT and 0.1% bromophenol blue), then boiled for 5 min. Equal amounts of protein were resolved on 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked at RT for 1 h in 5% (w/v) dry skim milk in TBS plus 0.1% Tween-20 (TBST), rinsed in TBST and incubated with primary antibodies at 4°C overnight. The primary antibodies used were mouse antibodies specific for β-actin, βIII-tubulin, GFAP, caspase-3 DCX, p62, Atg7, LC3B and CDK2 (all dilution, 1:1,000). After rinsing, blots were incubated in TBST with peroxidase-conjugated secondary antibodies at RT for 1 h. The secondary antibodies used included goat anti-mouse IgG and goat anti-rabbit IgG (both dilution, 1:1,000). The peroxidase reaction was visual-ized with an enhanced chemiluminescence reagent. Films were digitized and densitometry was performed using ImageJ software.

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