Phospho jak2 mab

Cell were lysed in CelLytic lysis buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Sigma-Aldrich) and a phosphatase inhibitor cocktail (Sigma-Aldrich). The protein concentration of cell lysates was measured using a BCA protein assay (Thermo Scientific). Five micrograms of protein was separated on 4–12% NuPage Bis-Tris gels (Invitrogen) and transferred to a polyvinylidene fluoride (PVDF) membrane using the iBlot Dry Blotting System (Invitrogen), according to the manufacturer's protocol. Blots were blocked in PBS (−) with 0.05% Tween 20 (Sigma-Aldrich) buffer (PBST) with 2% nonfat dry milk, followed by incubation overnight in PBST with 2% nonfat dry milk and primary antibodies. The following were used as primary antibodies and purchased from Cell Signalling Technology: JAK2 mAb, phospho-JAK2 mAb, STAT1 mAb, phospho-STAT1 mAb, p44/42 MAPK (Erk1/2) mAb, phospho-p44/42 MAPK (Erk1/2) mAb, and β-actin antibody. After washing in PBST, membranes were incubated with the HRP-linked anti-rabbit IgG (Cell Signalling Technology). Blots were visualized by ECL Prime (Amersham Pharmacia, Uppsala, Sweden) according to the manufacturer's protocol and with a film processor (Konica SRX-101A; JZ Imaging & Consulting, Willoughby, OH, USA).

The following were used as primary antibodies and purchased from Cell Signaling Technology: JAK2 mAb, phospho‐JAK2 mAb, STAT1 mAb, phospho‐STAT1 mAb, p44/42 MAPK (ERK1/2) mAb, phospho‐p44/42 MAPK (ERK1/2) mAb and β‐actin antibody. An HRP‐linked anti‐rabbit IgG (Cell Signaling Technology) was used as secondary antibody. All samples were prepared and stained with antibodies as previously described,8 and blots were visualized by ECL Prime (Amersham Pharmacia, Uppsala, Sweden) and with a Konica SRX‐101A film processor (JZ Imaging & Consulting, Willoughby, OH, USA).