Lipofectamin and plus reagent

Cells (1 × 10⁷) were lysed in immunoprecipitation buffer (50 mmol/liter HEPES, pH 7.6, 150 mmol/liter NaCl, 5 mmol/liter EDTA, 0.1% Nonidet P-40). After centrifugation (10 min at 15,000 × g) to remove particulate material, the supernatant was incubated with each antibody (2 μg/ml) with constant agitation at 4°C. The immune complexes were precipitated with protein A/G-Sepharose (Sigma) and analyzed by Western blot. To examine the effect of GTGKT peptide on the binding of CAGE to GSK3β, biotin-GTGKT peptide (Peptron, Daejeon, Korea) was transfected with Lipofectamin and PlusTM reagent (Invitrogen, San Diego, CA). After incubation for 48 h, whole-cell extracts were incubated with anti-biotin antibody (2 μg/ml) for 12 h at 4°C and immune complexes were precipitated with streptavidin-linked agarose beads for 30 min at 4°C. After five washes with lysis buffer, the bound proteins were eluted by boiling in 2X Laemli SDS loading buffer and were then subjected to SDS-PAGE followed by Western blotting analysis with anti-CAGE antibody.

Peptides used in this study were commercially synthesized by Peptron Company (Daejon, Koea). The FITC-conjugated AQTGTGKT, and Biotin-AQTGTGKT peptide were purified by high performance liquid chromatography and their sequence and structure were confirmed by mass spectrometry. To examine whether AQTGTGKT peptide binds to CAGE or Beclin1, biotin-AQTGTGKT peptide (Peptron, Daejeon, Korea) was transfected with Lipofectamin and PlusTM reagent (Invitrogen, San Diego, CA). After incubation for 48 h, whole-cell extracts were incubated with anti-biotin antibody (2 μg/ml) for 12 h at 4°C and immune complexes were precipitated with streptavidin-linked agarose beads for 30 min at 4°C. After five washes with lysis buffer, the bound proteins were eluted by boiling in 2X Laemli SDS loading buffer and were then subjected to SDS-PAGE followed by immunoblotting analysis with anti-CAGE antibody or anti-Beclin1 antibody.