Rabbit monoclonal anti stat3

Immunoblotting was performed according to standard protocols (Life Technologies). Electrophoresis was run in 4–12% gradient NuPAGE Novex Bis-Tris Pre-Cast Gels (Life Technologies) and followed by protein transfer to a PDVF or nitrocellulose membrane (Life Technologies) according to standard protocols. Blocking was performed in PBS 1×, 0.2% Tween, 5% dry milk powder (Marvel). Membranes were stained with the primary and secondary antibodies diluted in the same blocking solution. Primary antibodies used were as follows: mouse monoclonal anti-β-actin, 1:20,000 (Abcam), rabbit anti-EphB1, 1:500 (Santa Cruz), rabbit anti-ephrin-B1, 1:1000 (Santa Cruz), mouse monoclonal anti-GFAP, 1:500 (Sigma-Aldrich), rabbit monoclonal anti-STAT3, 1:1000 (Cell Signalling), rabbit monoclonal anti-pSTAT3 (Tyr705), 1:1000 (Cell Signalling), rabbit anti-Trim30, 1:1000, (Novus). Detection was performed by exposing the membrane to the Amersham ECL Prime WB Detection Reagents (GE Healthcare).

Proteins were extracted from whole cell lysates and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to a polyvinylidene fluoride membrane. The following primary antibodies were used: rabbit polyclonal anti-IGFBP-5 (1:1500; Abcam, Cambridge, UK), rabbit polyclonal anti-proteoglycan (1:1200, Abcam), rabbit polyclonal anti-type I collagen (1:1500, Abcam), rabbit polyclonal anti-type V collagen (1:2000, Abcam), rabbit monoclonal anti-phosphorylated mTOR (p-mOTR, 1:1000, Cell Signaling Technology, Danvers, USA), rabbit monoclonal anti-mTOR (1:1000, Cell Signaling Technology), rabbit monoclonal anti-phosphorylated STAT3 (p-STAT3, 1:2000, Cell signaling Technology), rabbit monoclonal anti-STAT3 (1:1000, Cell Signaling Technology), and rabbit monoclonal anti-β-actin (1: 5000; Cell Signaling Technology). Membranes were then incubated with the horseradish peroxidase-conjugated secondary antibodies (1:4000; Abcam). The ECL western blot substrate kit was used to detect the respective bands (Abcam). The membranes were exposed using a ChemoDoc XRS detection system (Bio-Rad, Milan, Italy).

Cells grown and treated as indicated were collected and total protein was extracted. The following primary antibodies were used: rabbit monoclonal anti-α-actin (Abcam), monoclonal anti-COL1A1 (Millipore), rabbit monoclonal anti-phosphorylated SMAD2, rabbit monoclonal anti-phosphorylated SMAD3, rabbit monoclonal anti- SMAD2/3, rabbit monoclonal anti-ERK1/2, or rabbit monoclonal anti-phosphorylated ERK1/2, rabbit monoclonal anti-Akt, rabbit monoclonal anti-phosphorylated Akt, rabbit monoclonal anti-STAT3, or rabbit monoclonal anti-phosphorylated STAT3 (all from Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat-anti-rabbit antibody (Thermo Scientific) was used as a secondary antibody. The control for equal protein loading was assessed with an anti-β-actin antibody (Cell Signaling Technology, Inc.).