TWEAK expression in bone-related cell lines and the mouse bone tissue. (a) The indicated tissues were obtained from Balb/c mice, and RNA was extracted. Real-time PCR was then performed using specific primers for TWEAK and GAPDH. The ratio of each gene to that of GAPDH was calculated, and the value of 0.1 was assigned to the brain tissue. (b) RNA was extracted from MC3T3-E1, RAW264, ATDC5, and EL4 cells, and then real-time PCR was performed using specific primers for TWEAK and GAPDH. The ratio of each gene to that of GAPDH was calculated, and the value of 0.1 was assigned to MC3T3-E1 cells. (c) Immunohistochemical examination of the mouse bone tissue. Mouse tail bone tissues from Balb/c mice were stained with anti-TWEAK antibody. Representative images of weak enlargement (upper panel) and strong enlargement (lower panel) are shown. Positive staining is indicated as brown. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.