Mab3324

Tissue sections or primary cells were fixed and blocked with 5% Donkey serum (Sigma-Aldrich, USA). Samples were then incubated with primary antibodies at 4°C overnight. The following antibodies were used for staining: Bglap (Abcam, USA, ab93876, 5 ug/ml); Tppp3 (Abcam, USA, ab150998, 1:50); Mkx (Lifespan Biosciences Inc., USA, LS-B8063, 1 ug/ml); Gli1 (R&D, USA, MAB3324, 10 ug/ml); Gli2 (R&D, USA, AF3635, 5 ug/ml); pH3 (Santa Cruz, USA, sc-8656-R, 1:200); Donkey anti-rabbit IgG (H + L), Alexa Fluor 594 conjugate (Thermo Fisher Scientific, USA, A21207, 1:500); Donkey anti-rat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A21208, 1:500); Donkey anti-goat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A11055, 1:500); and Goat anti-rabbit IgG (H + L), Alexa Fluor 633 conjugate (Thermo Fisher Scientific, USA, A21072, 1:500). Samples were washed with PBS and incubated with secondary antibodies at room temperature for 1 hr. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, USA). Signals were detected under fluorescence microscopy. For TUNEL assay, procedures were followed as described by the manufacturer using the ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit (Millipore, USA, s7111).

For immunoblotting, cells were lysed in Laemmli buffer, and protein concentration was determined using Pierce BCA protein assay kit (Life Technologies). 20 μg of protein was loaded per sample and resolved on a 8% (wt/vol) polyacrylamide–SDS gel and transferred onto nitrocellulose membranes, which were immediately blocked with 3% (w/v) BSA for 1 h. Membranes were then probed with an antibody against CTSK (Millipore, MAB3324) or proMMP-9 (R&D systems, MAB9111). GAPDH (Santa Cruz Biotechnology, SC-25778) was used as a loading control. Blots were developed using peroxidase-conjugated secondary antibodies and chemiluminescence system (Thermo Scientific).