Anti goat secondary antibodies

Lipofectamine 2000 and horseradish peroxidase (HRP)-coupled secondary anti-mouse and anti-rabbit antibodies were from ThermoFisher Scientific (Waltham, MA). Cycloheximide and mouse anti-FLAG (M2) antibody were from Sigma-Aldrich (St Louis, MI). FLT3 ligand (FL) was from ORF Genetics (Reykjavik, Iceland). Rabbit anti-phospho-AKT (pSer473), mouse anti-phosphotyrosine (4G10), mouse anti-mono-ubiquitin antibodies were from Abcam (Cambridge, UK), Merck Millipore (Billerica, MA) and Covance Research Products (Princeton, NJ), respectively. Rabbit anti-phospho-ERK1/2 (pThr202/pThr204), goat anti-AKT, rabbit anti-ERK2 and anti-goat secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The phycoerythrin-labeled anti-FLT3, mouse anti-phospho-p38 and anti-p38 antibodies were from BD Transduction Laboratories (Franklin Lakes, NJ). Rabbit anti-FLT3 antibody was described previously [29 (link)].

Heat-denatured protein samples (20μg) were separated on a 10% SDS-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membranes 0.2μm (Sigma). Affinity-purified goat anti-mouse IRF1 antibody (catalogue #: AF4715) and rabbit anti-goat IgG secondary antibody (catalogue #: HAF017) were obtained from R&D Systems Europe Ltd., United Kingdom, while rabbit anti-human IRG1 antibody (catalogue #: HPA040143) and normal rabbit IgG (catalogue #: sc-2027) were purchased from Sigma and Santa Cruz Biotechnology, respectively. Goat anti-β-actin (catalogue #: sc-1616) and anti-goat secondary antibodies (catalogue #: RPN1025) were purchased from Santa Cruz Biotechnology and GE Healthcare, respectively. After blocking with 5% (wt/vol) dry milk in PBS, the membrane was incubated overnight at 4°C with primary antibody in 1% BSA/PBS (dilution 1:2500 for IRF1 and IRG1) on a rotating platform. After three washing steps with PBS containing 0.1% Tween-20, the membrane was incubated with secondary antibodies (dilution 1:5000) coupled to horseradish peroxidase and revealed by chemiluminescence using the Amersham ECL detection reagents (GE Healthcare) and ODISSEY imaging system.