Molecular Signaling Pathways in Mast Cells

MCs were starved overnight in RPMI 1640 supplemented with 5%FCS and 15 ng/ml recombinant murine IL-3 and then stimulated with IL-33 (100 ng/ml) and/or SCF (100 ng/ml) alone or in combination for 15 min at 37 °C. Cells were homogenized in fresh prepared RIPA lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, Triton X-100 1%, SDS 0.1%, DOC 0.5%, EDTA 1 mM, EGTA 1 mM, MgCl2 5 mM, leupeptin 5μg/ml, PMSF 1 mM, aprotinin 5μg/ml, NaF 5 mM, Na₃VO₄ 1 mM). Lysates were centrifugated at 13,000 g for 20 min to eliminate debris, resolved by SDS-polyacrylamide gel (PAGE) and proteins were electro-blotted, as previously described [70 (link)].
Immunoreactive bands were visualized on the nitrocellulose membranes, using horseradish-peroxidase-linked/coupled donkey anti-rabbit or sheep anti-mouse IgG (GE Healthcare, Chicago, IL) and the Clarity max, western ECL substrate (Biorad, Hercules, CA) based on manufacturer’s instructions and exposed to Invitrogen iBrightTM Imaging Systems (Thermo Scientific).
The following Abs were used: anti-pSTAT3, anti-STAT3, anti-pAkt S473/D9E, anti-Akt, anti-pp38 T880/Y182, anti-p38, anti-pp44/42 (ERK1/2) (T202/Y204) and anti-P44/42 (ERK1/2) (all from Cell Signaling, Danvers, MA); anti-Ikb-α (Santa Cruz Biotechnologies, Dallas, TX); anti-actin (Merck Life Sciences). Densitometric analysis was performed using FIJI Image J software.