Frozen tissues were homogenized in TRIzol (Invitrogen, Carlsbad, CA) and cDNA prepared as described [47, 48] . Quantitative real-time PCR was performed using 4 ml of cDNA and SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in duplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems). Expression levels were normalized to ubiquitin or GAPDH using the following formula: 