Complete inhibitor

Isolated TECs were lysed in a buffer containing 1× TBS, 0.01% NP-40, 200 μM PMSF, cOmplete inhibitor (Roche, 11697498001), 50 mM sodium fluoride, 2 mM sodium orthovanadate, and 30 mM sodium pyrophosphate. Immunoprecipitation was performed with 0.14 μg HSP70 antibody (CST, 4872) overnight at 4°C rotating. Protein G beads (MilliporeSigma, P3296) were added for 3 hours at 4°C rotating before 3× sample buffer was added and samples were boiled at 95°C for 10 minutes. Supernatants were analyzed by Western blot.

Western blotting was performed as described.¹⁴ Cultured cells (1 × 10⁶) were lysed in 500 µL lysis buffer (20 mM Tris‐HCl, 150 mM NaCl, 1% Triton X‐100, 1 mM EDTA, 1 mM Na2VO4, 1 mM PMSF, 10 mM 1,10‐phenanthroline monohydrate) supplemented with 1‐fold Complete Inhibitor (Roche), incubated for 10 minutes and cleared by centrifugation at 16 000 g for 5 minutes. Cell lysates containing 15‐20 µg protein (according to bicinchoninic acid assay, Thermo Fisher/Pierce) were heated in SDS reducing sample buffer (250 mM Tris‐HCl (pH 6.8), 50% (w/v) glycerol, 10% (w/v) SDS, 0.1% bromophenol blue and 5% β‐mercaptoethanol) and subjected to SDS‐polyacrylamide gel electrophoresis using 10% Tris‐glycine gels. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond‐P, Amersham; 10 600 022). Membranes were blocked with 5% (w/v) non‐fat dry milk in Tris‐buffered saline with 0.05% Tween. For analysis of phosphoproteins, dry milk was substituted by 5% bovine serum albumin (BSA). Membranes were probed overnight with primary antibodies followed by incubation with POD‐coupled secondary antibodies (diluted 1:20 000). After addition of chemiluminescence substrate (ECL advanced, Amersham), signals were recorded using luminescent image analyser LAS2000 (Fujifilm, Tokyo, Japan) and quantified using open source ImageJ software (Wayne Rasband, NIH).

Cells were collected by trypsin-EDTA and dissolved in NP40 lysis buffer (0.5% NP40, 250mM NaCl, 50mM Hepes, 5mM EDTA and 0.5mM EGTA) freshly supplemented with Complete inhibitor (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail 1&2 (Roche, Mannheim, Germany). The following antibodies were used for the detection of p53 (polyclonal p53 Antibody #9282 Cell Signaling, Danvers, MA, 1:1000), CDKN1A/p21 (clone CP74, P1484 Sigma-Aldrich, St. Louis, MO, 1:200), PUMA (polyclonal PUMA Antibody #4976 Cell Signaling, Danvers, MA, 1:1000) and β-actin (clone AC-40, A4700 Sigma Aldrich, St. Louis, MO, 1:1000).

Cells in 6-well plates and frozen tumor samples were lysed by adding appropriate volumes of RIPA buffer (Thermo Scientific) supplemented with complete inhibitor (Roche), Halt protease inhibitor and Halt phosphatase inhibitor (both from Thermo Scientific). The lysate was collected in a 1.5 mL tube and further processed as described in the Suppl. Information.

The level of protein S-palmitoylation was assessed as described (Yokoi et al., 2016 (link)), with minor modifications. HeLa cells were lysed with the following buffer (4% SDS, 5 mM EDTA, in PBS with complete inhibitor (Roche)). After centrifugation at 100,000 × g for 15 min, supernatant proteins were reduced with 25 mM TCEP for 1 hr at 55°C or at room temperature (RT), and free cysteine residues were alkylated with 20 mM NEM for 3 hr at RT to be blocked. After chloroform/methanol precipitation, resuspended proteins in PBS with 4% SDS and 5 mM EDTA were incubated in buffer (1% SDS, 5 mM EDTA, 1 M NH₂OH, pH 7.0) for 1 hr at 37°C to cleave palmitoylation thioester bonds. As a negative control, 1 M Tris-HCl, pH7.0, was used. After precipitation, resuspended proteins in PBS with 4% SDS were PEGylated with 20 mM mPEGs for 1 hr at RT to label newly exposed cysteinyl thiols. As a negative control, 20 mM NEM was used instead of mPEG (-PEG). After precipitation, proteins were resuspended with SDS-sample buffer and boiled at 95°C for 5 min. Protein concentration was measured by BCA protein assay.