Ab75603

Cultured cells were washed in cold phosphate-buffered saline (PBS), lysed in 100mM Tris-HCl pH 8.0, 2% SDS supplemented with protease inhibitors (Roche), rotated for 20 min then centrifuged at 13,000 g. After centrifugation, the supernatant was mixed with 2x Laemmli Buffer, heated for 5 min at 95°C, and electrophoresed on a NuPAGE 4–12% Bis-Tris gradient gel. Mouse tissue lysates were similarly prepared but with Dounce homogenization. Following electrophoresis, gels were transferred onto PVDF membranes and probed with rabbit anti-MeCP2 (1:3,000, Zoghbi lab, #0535) (69 (link)), rabbit anti-GST (1:2000, Sigma-Aldrich G7781), rabbit anti-HIPK2 pY361 (1:500, Invitrogen), rabbit anti-MeCP2 pS80 (1:500, Active Motif), rabbit anti-PPP2R1A (1:3,000, Abcam, ab154551), mouse anti-Tau (1:2500, Abcam ab80579), rabbit anti-Tau pS356 (1:1000, Abcam ab75603), mouse anti-GAPDH 6C5 (1:20,000, Advanced Immunochemicals, 2-RGM2), and mouse anti-Vinculin (1:10,000, Sigma-Aldrich). Secondary antibodies were mouse anti-rabbit horseradish peroxidase (HRP) (1:3,000, Jackson ImmunoResearch Labs, 211-032-171) and donkey anti-mouse HRP (1:50,000, Jackson ImmunoResearch Labs, 715-035-150). Immunoblot images were acquired with the ImageQuant LAS 4000 (GE Healthcare) and quantified with ImageJ.