Western blot analysis
Primary Th2 cells were harvested in lysis buffer and protein
electrophoresed on SDS-Page gel. Gels were wet transferred onto
nitrocellulose membrane, blocked with milk (Carnation skim milk powder,
5%) and incubated with antibody against CRTh2. Membranes were washed in
TBS-T and incubated with horseradish peroxidase secondary antibody.
Bands were detected using Clarity Western ECL substrate (Bio-Rad, CA,
USA) and images were acquired using a ChemiDoc MP Imaging System. GAPDH
was detected after stripping the membranes and re-probing with antibody
against GAPDH. Densitometry of CRTh2 was normalized to GAPDH using
ImageJ.