Sesn2

pCMV6-Alox5-GFP (Origene) was transfected into 293T cells with vectors expressing FLAG-tagged MAPKs. The cells were lysed 24 h later and immunoprecipitated with anti-GFP (Origene), followed by Western blot with anti-phospho-Alox5^S217 (Cell Signaling Technology, Danvers, MA, USA). The blots were also probed with anti-GFP. Total lysates were used for Western blot by probing with anti-FLAG (Sigma, St. Louis, MO, USA). JMR cells transfected with siRNA targeting specific genes or non-targeting siRNA control were lysed for Western blot by probing with specific antibodies. The following antibodies were used for Western blot: rabbit polyclonal or monoclonal antibodies to Alox5, ERK1, p38 MAPK, p44/42 MAPK (ERK1/2), phosphor-p44/p42 MAPK (p-ERK1/ERK2), RAD51, SAPK/JNK, SESN2 (Cell Signaling Technology), Alox5AP, DHODH, HADHA, MGST1, TIA-1 (Abcam), EndoG (ProSci), OXR1 (Bethyl Laboratory, Montgomery, TX, USA.) and PDP1 (Sigma); and mouse monoclonal antibodies to AIFM1, API5, PNKP (Santa Cruz Biotechnology), caspase-3, caspase-6 caspase-7, caspse-9, phosphor-p38 MAPK, phosphor-SPAK/JNK (Cell Signaling Technology), FLAG (Sigma) and GFP (Origene). The blots were also probed with mouse monoclonal anti-α tubulin (Santa Cruz Biotechnology) to ensure equal loading.

Whole-cell lysates were prepared with a lysis buffer and protein concentration of each sample was measured using a DC Protein Assay Kit (BIO-RAD Laboratories, Madison, WI). After normalization, equal amount of protein was separated by SDS-PAGE and transferred to Immuno-Blot PVDF membranes. The membranes were blocked with 5% skim milk in TBST at RT for 2 h and incubated with primary antibodies and corresponding HRP-conjugated secondary antibodies. Antibodies against cleaved PARP, cleaved caspase-3, SESN2, p-mTOR, mTOR, and Mcl-1 were purchased from Cell Signaling Technology, Inc. (Charlottesville, VA) and actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The immunoreactive bands were visualized by ImageQuant LAS 500 (GE Healthcare Life Sciences, Piscataway, NJ).

S6K1, phospho-S6K1(Thr389), rpS6, phospho-rpS6(Ser235/236), IRE1α, CHOP, GADD34, ATF4, TSC2, SESN2, AMPK, phospho-AMPK(Thr172), ACC, phospho-ACC (Ser79) and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). LC3 antibodies were bought from Novus Ltd. (Cambridge, UK). Cells were washed in ice-cold phosphate buffered saline (PBS) and then lysed in cell lysis buffer (20 mM Tris (pH 7.5), 125 mM NaCl, 50 mM NaF, 5% (v/v) glycerol, 0.1% (v/v) Triton X-100, supplemented with 1 mM dithiothreitol (DTT), 1 µg/mL pepstatin, 20 µM leupeptin, 1 mM benzamidine, 2 µM antipain, 0.1 mM PMSF, 1 mM sodium orthovanadate and 1 nM okadaic acid prior to cell lysis). Cell lysates were sonicated using a diagenode bioruptor (Diagenode, Seraing, Belgium) and centrifuged at 13,000 rpm for 8 min. Protein concentrations were determined by a Bradford assay (Thermo Fisher Scientific, Paisley, UK). Samples were diluted in 4 × NuPAGE loading sample buffer (Life Technologies) with 25 mM DTT and boiled at 70 °C for 10 min. Western blot was performed as previously described [34 (link)].