Pstat5 9351

Cells were serum starved for 16 hours in Roswell Park Memorial Institute medium (RPMI) with 0.7% fetal calf serum (FCS) before inhibitor treatment. Cell lysates in RIPA buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxylate, 1% nonyl phenoxypolyethoxylethanol [NP40]) were subjected to western blotting with the following antibodies, all from Cell Signaling Technologies (Danvers, MA, USA): pIGF-1R (3021) (phosphorylated insulin like growth factor-1-receptor); pJAK2 (3771) (phosphorylated janus kinase 2); pSTAT5 (9351) (phosphorylated signal transducer and activator of transcription 5); pMEK (9121) (phosphorylated MAPK/ERK kinase); pERK (4370) (phosphorylated extracellular signal regulated kinase); pAKT (9275) (phosphorylated protein kinase B); IGF-1R (3027); JAK2 (3230); STAT5 (9352); MEK (9122); ERK (9102); AKT (9272). Blots were stripped and reprobed against beta actin (Sigma A5441) to ensure equal loading. Immune complexes were detected using chemiluminescence (K12-045-D20, Advansta, San Jose, CA, USA).

Protein extracts separated on SDS-polyacrylamide gel electrophoresis, were blotted on nitrocellulose membranes and probed with antibodies against JAK2 (D2E12 #3230), pSTAT5 (#9351), Ezh2 (Ac22 #3147), SMAD2/3 (#5339), p38 (#9213) and p-p38 (T180/Y182 #4511) (all from Cell Signaling, Boston, MA, USA), STAT5 (#sc-835, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (#CB1001, Calbiochem, San Diego, CA, USA).