Rabbit anti snap25

For total protein extraction, samples were homogenized using the following lysis buffer composed by 1% sodium dodecyl sulfate (SDS), 10mM HEPES at pH 7.4 and 2 mM EGTA and protein concentration was estimated using Bicinchoninic Acid Assay (BCA) kit (Thermo Fischer Scientific) as 20μg. Proteins were loaded with 2x Loading Buffer (100 mM Tris-HCl at pH 6.8; 4% SDS; 20% Glycerol; 200 mM 2-Mercaptoethanol, 2 mg Bromophenol-Blue) and fractionated by SDS-PAGE, then transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). Membranes were stained with the following primary antibodies: mouse anti-STAT3 (Cell Signaling, 1:1000), rabbit anti-STAT3 P-Tyrosine 705 (Cell Signaling, 1:1000), mouse anti-STAT3 P-Serine 727 (Cell Signaling, 1:1000), rabbit anti-GAPDH (Synaptic System, 1:4000), mouse anti-PSD95 (UC Davis/NIH NeuroMab Facility, CA; 1:1000), guinea pig anti-VGLUT1 (Synaptic System, 1:1000), rabbit anti-Shank2/3 (Synaptic System, 1:1000), mouse anti-GAP43 (Millipore, 1:1000), rabbit anti-NFKB (Cell Signaling; 1:1000), rabbit anti-SNAP25 (Synaptic System, 1:1000), mouse anti-p65 (Cell Signaling; 1:1000). Immunodetection was performed with Clarity Western ECL Substrate (Bio Rab) and analyzed through Chemidoc apparatus via ImageLab software (Bio-Rab).