Effect of preconditioning with TWEAK on cerebral ischemia-induced cell death. (A) Representative micrographs of Wt cerebral cortical neurons immunostained for pBAD 3 hours after 60 minutes of treatment with either vehicle (control; panels a-c) or TWEAK 100 ng/mL (panels d-f). Green is pBAD, blue is DAPI. Magnification × 40. (B) Representative western blot analysis for pBAD and total BAD expression 0 to 180 minutes after 60 minutes of incubation with TWEAK alone or in combination with SL327 10 μM. (C) Representative micrographs of TUNEL staining in area of AOI2 in Wt mice intraperitoneally injected with saline solution (panels a and b), TWEAK 0.2 mg (panels b and c), or with a combination of TWEAK and SL327 (panels c and f) 24 hours before tMCAO. Green is TUNEL, blue is DAPI. Magnification × 20. (D) Diagram showing the three different AOIs and percentage of TUNEL-positive cells per field AOI 1, 2 and 3 in the ischemic tissue of Wt mice intraperitoneally injected with saline solution (black bar), TWEAK 0.2 mg (white bar), or TWEAK plus SL327 24 hours before tMCAO. n = 10, *P < 0.05 compared to untreated mice. Lines depict SD. AOI: area of interest; BAD: Bcl-2-associated death promoter protein; DAPI: 4'-6-diamidino-2-phenylindole; pBAD: BAD phosphorylated at Ser112; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick end labeling; TWEAK: tumor necrosis factor-like weak inducer of apoptosis.