Phospho erk1 2

Akt, ERK, and FOXO activities were determined as described previously^{38 (link)}. Briefly, PC12 parental and PC12 mutant cells were plated at a density of 1 × 10⁶ cells/25 cm² in a flask of serum-containing medium and cultured for 3 days. Then the culture medium was replaced with medium containing 0.5% FBS, and the cells were cultured for a further 48 h. PC12 parental, PC12m3 and PC12m321 cells were then treated for 10 or 30 min with NGF (30 ng/ml) or exposed to heat shock (44 °C) for 30 min. Akt, ERK, and FOXO activities in cell lysates were then assayed. The cells were lysed in a lysing buffer. Aliquots of the lysates (10–15 μg) from each sample were fractionated on an SDS-10% polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The blots were probed with antibodies specific for phospho-Akt, phospho-ERK1/2, phospho-FOXO, total Akt, or total FOXO (New England BioLabs; Beverly, MA) at a dilution of 1:1000 in blocking buffer (5% nonfat dry milk in PBS) for 12 h at 4 °C. The blots were probed with a secondary antibody, horseradish peroxidase-linked anti-rabbit IgG, at a dilution of 1:2000 in blocking buffer for 60 min at 25 °C. The blots were stained for 1 min using a nucleic acid chemiluminescence reagent (LumiGLO chemiluminescent reagent, Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) and exposed to x-ray film.

Neutrophils were harvested and analyzed by Western blot using polyclonal antibodies to AKT, phospho-AKT, phospho-ERK1/2, IκB, Mcl-1, Bax, and β-actin (New England Biolabs, Hertfordshire, UK). Detection was performed with the appropriate peroxidase-conjugated secondary antibodies (1:2,000) for 1 h. Chemiluminescence (ECL-plus-kit GE Life Sciences, Buckinghamshire, UK) and exposure to X-ray film (XOMAT-AR; GE life sciences, Buckinghamshire, UK) were used to detect banding.