Anti-p62 (SQSTM1) (Human) pAb

Protocol tips

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	Dilute Primary Ab at 1∶2000 and incubate for 1hr at RT

Protocol tips
Dilute Primary Ab at 1∶2000 and incubate for 1hr at RT

Publication protocol

SH-SY5Y cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% IGEPAL and 0.5% sodium deoxycholate) containing a protease inhibitor cocktail (code P8349; Sigma-Aldrich). For detection of phosphoproteins, SH-SY5Y cells were lysed in ice-cold buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton, 1 nM okadaic acid, protease (code P8349, Sigma-Aldrich) and phosphatase (code 524625, Calbiochem) inhibitor cocktails.

Lysates were centrifuged for 15 min at 20.000 g at 4°C and supernatants were assayed for protein content by Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Milan, Italy). Equal amount of total proteins was resolved by 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Immobilon-P, Sigma-Aldrich). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized with the ECL Western Blotting Detection kit (ECL, Amersham Biosciences, GE Healthcare, Milan, Italy), and the chemiluminescence signal detected using X-ray films (Hyperfilm ECL, Amersham Biosciences). Exposure time to obtain the optimal density of the images varied from 5 s to 20 min. Autoradiographic films were scanned, digitalized at 300 dpi and band quantification was performed using ImageJ software (NIH, Bethesda, MD, USA).

The following primary antibodies and dilutions were used: anti-LC3 1∶1000 (code PD036; MBL), anti-p62/SQSTM1 1∶2000 (code PM045; MBL), anti-Beclin-1 1∶2000 (code PD017; MBL), anti-phospho p70S6K (Thr389) 1∶1000 (code 9234; Cell Signalling Technology), anti-p70S6K 1∶2000 (code 2708; Cell Signalling Technology), anti-phospho ULK (Ser757) 1∶1000 (code 6888; Cell Signalling Technology), anti-ULK 1∶1000 (code 8054, Cell Signalling Technology), anti-actin 1∶1000 (clone AC-40; Sigma-Aldrich), anti-β-tubulin 1∶30000 (clone B-5-1-2; Sigma-Aldrich), anti-GAPDH 1∶40000 (clone 6C5; Applied Biosystems, Carlsbad, CA, USA). HRP-conjugated goat anti-rabbit or anti-mouse IgG (Pierce Biotechnology, Rockford, IL, U.S.A) were used as secondary antibodies.

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