Protease phosphatase inhibitor cocktail

Cells were lysed in 1% Triton-X100, 20 mM HEPES-NaOH (pH 7.5), 500 mM NaCl, and protease/phosphatase inhibitor cocktail (Nacalai Tesque Inc., Kyoto, Japan), and the soluble fractions were collected by centrifugation at 20000 × g for 15 min at 4°C. Fractions were analyzed by SDS-PAGE, and proteins in the gel were transferred to an Immobilon-P PVDF membrane (EMD Millipore, Billerica, MA, USA). Proteins were detected with a mouse monoclonal antivimentin antibody (V9, Sigma) and anti-β-actin antibody (Sigma). Detection was performed using a C-DiGit® Blot Scanner (LI-COR Inc., Lincoln, NE, USA). Blotting data were processed using Image Studio Lite software (LI-COR).

Cells were lysed with radioimmunoprecipitation assay buffer (Fujifilm Wako Pure Chemical) containing protease/phosphatase inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were transferred onto polyvinylidene difluoride membranes. After blocking, the blots were incubated with the following antibodies: anti‐p21^Cip1/Waf1 (#2947; Cell Signaling Technology [CST]), anti‐p16^Ink4a (SPC‐1280; StressMarq Biosciences), anti‐γH2AX(Ser¹³⁹) (#9718; CST), anti‐Bcl‐2 (#658701; BioLegend), anti‐Bcl‐xL (#2764; CST), anti‐Mcl‐1 (#54535; CST), anti‐survivin (#71G4B7; CST), anti‐cFLIP (ALX‐804–428; Enzo Life Sciences), anti‐TATA‐binding protein (TBP; #22006‐I‐AP; Proteintech), anti‐PARP (#46D11; CST), anti‐caspase‐3 (#9668; CST), anti‐c‐Myc (1472–1; EPT), anti‐GAPDH (#015‐25473; Fujifilm Wako Pure Chemical), and anti‐β‐actin (#622102; BioLegend). Nuclear and cytoplasmic proteins were prepared using the LysoPure™ Nuclear and Cytoplasmic Extraction Kit (Fujifilm Wako Pure Chemical). After washing, membranes were incubated with goat anti‐rabbit or horse anti‐mouse horseradish peroxidase‐conjugated secondary antibody (#7074 and #7076; CST). Protein bands were visualized using an Amersham ImageQuant™ 800 Biomolecular Imager (General Electric Company).

HEK293 cells were cultured in the presence of 1 μg ml⁻¹ doxycycline and 1 μM epoxomicin, with or without 10 μM FINDY for 8 h. The cells were then harvested and lysed in 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1% Empigen BB and protease/phosphatase inhibitor cocktails (Nacalai Tesque). The clarified cell lysates were processed with anti-FLAG (M2) beads (Sigma-Aldrich) and the bound proteins were then eluted with glycine buffer (pH 3.0), which was immediately neutralized with 1 M Tris (pH 8.0). After reductive alkylation, the samples were trypsinized, then processed using ZipTip pipette tips containing C18 (Millipore). The digested and purified peptides were analysed by LC-MS/MS on TripleTOF 5600 (AB SCIEX). Database searches were performed using ProteinPilot Software (AB SCIEX).