Phospho stat1 9167

Cell lysates (50 μg) were separated on 7.5% TGX gels, transferred onto polyvinyldifluoride membranes (Bio-Rad, UK) and blocked using 5% (wt/vol) skimmed milk in tris-buffered saline (TBS)/0.1% (vol/vol) tween-20 for 1 h at room temperature. Blots were incubated overnight at 4°C with primary Abs: phospho-p38 (4511), phospho-p65 NF-kB (3031), β-actin (12262), phospho-TBK1 (5483), phospho-IRF3 (4947) or phospho-STAT1 (9167) from Cell Signalling Technology (NEB, Herts, UK) (1:1000 dilution in TBS, 1% milk). After washing in TBS/0.1% (vol/vol) tween-20, blots were incubated with HRP-conjugated secondary Ab at room temperature for 1 h in TBS/0.1% (vol/vol) tween-20 and 5% milk. After the final wash, immunoreactivity was visualized using the chemiluminescent substrate ECL Plus (Bio-Rad, UK). G-Box imaging system and Genesys software (Synoptics UK) were used to visualize blots and densitometry analysis was performed using Genetools (Synoptics UK). The level of cellular beta actin was used as a loading control.

Cells were lysed directly into SDS sample buffer and aliquots run on 10% polyacrylamide gels using standard methods^{51 (link)}. Proteins were transferred onto nitrocellulose membranes, and specific proteins were detected by immunoblotting. Antibodies against tyrosine phosphorylated STATs were from Cell Signaling Technology (phospho-STAT1 #9167, phospho-STAT3 #9131, phospho-STAT5 #4322 and phospho-STAT6 #9361). The total ERK1/2 antibody used as a loading control was also from cell Signaling Technology (#4695). HRP-conjugated secondary antibodies were obtained from Pierce. Bands were detected using Clarity ECL reagents from BioRad and imaged on a Licor Odyssey Fc system. Quantification was carried out using Licor Image Studio software. After correction for the ERK1/2 levels to account for loading differences, percentage inhibition was calculated relative to the IL-4 stimulated condition. IC50 values were calculated using the same methods as for the in vitro IC50 values.