Protease inhibitor complex

Cells were washed twice in 1X PBS and collected by trypsinization. Trypsin was inactivated with complete growth media. Then, cells were centrifuged and washed twice in 1X PBS. The cell pellet was incubated for 10 min in 5 volumes of hypotonic buffer (10 mM Tris, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, and 1X protease inhibitor complex (Roche)), cells were then centrifuged, resuspended in 2 volumes of hypotonic buffer and finally lysed by mechanical homogenization. The supernatant was supplemented with additional 30 mM Tris pH 7.9, 140 mM KCl, and 3 mM MgCl₂, then it was cleared by centrifugation at 15,000 × g for 30 min at 4 °C and stored as a cytosolic extract. Nuclei were collected and washed 3 times in hypotonic buffer, then were sequentially resuspended in nuclear extraction buffers (20 mM Tris, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 1X protease inhibitor complex (Roche)), starting with 100 mM NaCl and increasing salt concentration by steps of 100 mM until 600 mM NaCl was reached. For each step, nuclei were incubated for 7 min at 4 °C and then centrifuged at 4000 × g for 5 additional minutes. Supernatants were collected and cleared by centrifugation at 15,000 × g for 30 min at 4 °C. Fractions were analyzed by western blot, loading equal volumes of each one.

Cells were lysed in buffer (50 mM Tris–HCL [pH 7.4], 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 250 mM mannitol, 1% [v/v] Triton X-100) containing protease inhibitor complex (Roche) and phosphatase inhibitor (Life Technologies). Equal amounts of protein were loaded on precast NuPAGE Bis-Tris Gels (Life Technologies) followed by transfer onto nitrocellulose. For co-immunoprecipitation, one 15-cm plate containing 80% cell confluency was used for each experimental condition. Cells lysate was scrapped with lysis buffer (150 mM KCl, 0.2% [v/v] NP-40, 10% [v/v] glycerol, 20 mM Tris at pH 7.5, 0.5 mM DTT) with protease inhibitor complex (Roche) and phosphatase inhibitor (Life Technologies) on ice. Equal amounts of lysate (500 μg to 1 mg) were immunoprecipitated with 4 μg of anti-p53 (Santa Cruz). Protein G agarose or anti-Flag conjugated agarose beads (20 μl) were added to each tube and rotated at 4˚C for 24 h. Beads were washed four times with lysis buffer and resuspended in SDS loading dye and boiled for immunoblotting.

Cells grown in 6 cm dish were lysed on ice in lysis buffer (50 mM Tris-HCL [pH 7.4], 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 250 mM mannitol, 1% [v/v] Triton X-100) containing protease inhibitor complex (04693159001, Roche). The protein concentrations of the lysates were measured using the BCA Assay kit (23225, Thermo). The lysates were boiled with NuPAGE LDS-PAGE sample buffer (1771559, Invitrogen) supplemented with 5% β-mercaptoethanol (M3148, Sigma) for 5 min. Equal amounts of protein were loaded on precast NuPAGE Bis-Tris Gels (NP0321BOX, Life Technologies) followed by transfer onto nitrocellulose membrane (1620115, Bio-Rad). The immunoblotting was performed with the following antibodies: anti-PFK1 (1:1000) (ab154804, Abcam), anti-β-Actin (1:5000) (A1978, Sigma), anti-caspase 3 (1:1000) (9665s, Cell signaling), anti-cleaved-caspase 3 (1:1000) (9664s, Cell Signaling), anti-p53 (1:200) (sc-126, Santa Cruz), anti-phospho p53 (1:1000) (9284, Cell Signaling), anti-p53 (1:1000) (32532, Cell Signaling).