LC3B Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lyse cells in uffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitors	Dilute primary Ab at 1:5,000 and incubate overnight at 4C in a humidified container

Upstream tips
Lyse cells in uffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitors
Protocol tips
Dilute primary Ab at 1:5,000 and incubate overnight at 4C in a humidified container

Publication protocol

Total cell and nuclear lysates were prepared as described previously [21]. Briefly, for total cell lysate preparation, control and WSPIS-treated cells were collected by centrifugation and then the pellets were suspended at 4 °C for 1 h in a lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitors. For nuclear lysate preparation, cells were suspended in hypotonic buffer (10 mM HEPES (pH 7.5), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM NaF, 1 mM DTT, 1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride) and on ice for 15 min. Vigorously vortexed after 0.5% Nonidet P-40 was added. The nuclei were pelleted and resuspended in high-salt buffer (20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM phenylmethanesulfonyl fluoride). The nuclear lysates were centrifuged and the supernatants were frozen at −80 °C. For Western blotting, equal amounts of proteins were resolved on 12% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated with the appropriate primary antibodies in blocking buffer. Detection was performed with an enhanced chemiluminescence system and exposed to X-ray films.

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