Stat 1 tyr701

Phospho-specific and pan antibodies against JAK-1 (Tyr1022/1023), JAK-2 (Tyr1007/1008), STAT-1 (Tyr701) and STAT-3 (Tyr705) were purchased from Cell Signaling Technology (Beverly, MA).

Following treatment, microglial culture media were removed and cells were lysed with the 2x Laemmli sample buffer containing 5% 2-mercaptoethanol. The samples were then heated at 100°C for 5 min and vortexed before being loaded onto 4–15% precast acrylamide gels for electrophoresis. Gels were transblotted onto nitrocellulose membranes (0.45 µm), rinsed in TTBS (Tris-HCl with NaCl and Tween 20), and blocked with 5% Blotto (Santa Cruz, Dallas, TX) for 1 h at RT. Membranes were then probed with rabbit anti-β-actin (#4970) and rabbit anti-phospho-Stat3_(Tyr705) (#9131) or -Stat1_(Tyr701) (#9171) Abs (Cell Signaling Technology, Danvers, MA) overnight at 4°C. After washing 3x with TTBS, alkaline phosphatase (AP) conjugated secondary antibody (1 : 5,000 in 1% Blotto, Promega, Madison, WI) was added to the membranes at RT for 1 h. Following 3x washing with TTBS, membranes were rinsed in the assay buffer (1x) twice (2 min each) and then incubated in the substrate solution (CDP-Star, Applied Biosystems, Foster City, CA) for 10 min followed by imaging using an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE).