Figure 5. Acp5, Ctsk and Calcr mRNA levels are decreased in Lyz2^Cre;Nfatc2^Δ/Δ Osteoclasts.

BMMs derived from 1 month old Lyz2^Cre/WT;Nfatc2^Δ/Δ mice and Nfatc2^loxP/loxP control littermates were cultured for 4 days in the presence of M-CSF at 30 ng/ml and of RANKL at 10 ng/ml. The cells were collected at the indicated times for extraction of total RNA and proteins. (A) Nfatc2 and Nfatc1 mRNA levels were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels. Values are means ± SD; n = 5 control and n = 3 Nfatc2^Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2^Δ/Δ and control, p < 0.05. (B) 50 μg of total protein were separated by SDS-PAGE and NFATc1 and NFATc2 levels were detected by using immunoblot anti-NFATc1 and anti-NFATc2 antibodies, respectively. β-Actin levels served as a loading control in the same blot. (C) Tnfrs11a, Acp5, Ctsk and Calcr mRNA levels in total RNA were measured by qRT-PCR. Transcript levels are reported as copy number corrected for Rpl38. Values are means ± SD; n = 5 control and n = 3 Nfatc2^Δ/Δ biological replicates. Two technical replicates were used for each qRT-PCR reaction. *Significantly different between Nfatc2^Δ/Δ and control, p < 0.05.