To evaluate the effects of PGDHC on the promotion of CatB and CatK activities in vivo, a mouse model of calvarial injection was employed with some modifications (Kanzaki et al, 2017). Briefly, wild type male C57BL/6 mice were anesthetized with isoflurane (Patterson Veterinary, Cat# NDC14043‐704–06), after which they received a 100 μl intra‐periosteal injection of either of the following solutions: 1) PBS (Gibco, Cat# 10010049); 2) 10 μg/ml of recombinant RANKL (Biolegend, Cat#769406) in PBS; or 3) a mixture of 10 μg/ml of recombinant RANKL and 10 μg/ml of PGDHC in PBS. The injections were repeated every other day for 10 days.
The mouse calvaria from the calvaria injection experiments were carefully dissected and homogenized in Trizol (Invitrogen) followed by RNA purification using the RNeasy Micro Kit (Quiagen) and qRT‐PCR was performed as described above.
The mouse calvaria from the calvaria injection experiments were carefully dissected and homogenized in Trizol (Invitrogen) followed by RNA purification using the RNeasy Micro Kit (Quiagen) and qRT‐PCR was performed as described above.