Figure 7
Figure 7. Sbds ablation is associated with impaired RANK signaling. (A) Signaling downstream of RANK was evaluated by immunostaining for NF-κB on day 6 of OCG. NF-κB was found to be activated only in WT osteoclasts as shown by its exclusively nuclear distribution (arrows). Sbds-null osteoclasts did not exhibit active NF-κB as shown by its cytoplasmic distribution. Both Sbds-null and WT osteoclasts demonstrated activated, nuclear c-Jun, a transcription factor not directly involved in RANK signaling in osteoclasts. The staining pattern was identical in day 4 osteoclasts. (B) Alternatively, activation of NF-κB was measured by performing an ELISA on NF-κB content in nuclear extracts of day 4 osteoclasts. Stimulation of WT cells by M-CSF and RANKL resulted in a significant 5.8-fold increase in nuclear NF-κB signal versus stimulation by M-CSF alone (n = 4): *P < .005. There was an insignificant increase in nuclear NF-κB signal after stimulation of Sbds-null cells with M-CSF and RANKL: P = .065. (C) Specificity of the NF-κB ELISA was verified with the addition of WT or mutant oligonucleotide competitors. WT oligo, representing the native consensus binding sequence of NF-κB (5′-GGGACTTTCC-3′), significantly inhibited NF-κB binding in the positive control (Jurkat cells stimulated with PMA and A23187), WT and Sbds-null nuclear extracts, whereas the mutant consensus oligonucleotide had no effect. Specificity of 1 signifies 100% specificity (n = 4): *P < .01. (D) Translocation of NFATc1 into the nucleus was evaluated as a second marker of the RANK signaling pathway by performing an ELISA on NFATc1 content in day 4 nuclear extracts. Stimulation of WT cells by M-CSF and RANKL resulted in a significant 2.5-fold increase in nuclear NFATc1 signal versus stimulation by M-CSF alone. There was no increase in nuclear NFATc1 signal after stimulation of Sbds-null cells with M-CSF and RANKL (n = 4): *P < .005. (E) NFATc1 ELISA showed good specificity as the WT oligo, representing the native consensus binding sequence of NFATc1 (5′-AGGAAA-3′), significantly inhibited NFATc1 binding in the positive control (leukoagglutinin-stimulated Jurkat cells), WT and Sbds-null nuclear extracts, whereas the mutant consensus oligonucleotide had no effect (n = 4): *P < .005; **P < .01.

Sbds ablation is associated with impaired RANK signaling. (A) Signaling downstream of RANK was evaluated by immunostaining for NF-κB on day 6 of OCG. NF-κB was found to be activated only in WT osteoclasts as shown by its exclusively nuclear distribution (arrows). Sbds-null osteoclasts did not exhibit active NF-κB as shown by its cytoplasmic distribution. Both Sbds-null and WT osteoclasts demonstrated activated, nuclear c-Jun, a transcription factor not directly involved in RANK signaling in osteoclasts. The staining pattern was identical in day 4 osteoclasts. (B) Alternatively, activation of NF-κB was measured by performing an ELISA on NF-κB content in nuclear extracts of day 4 osteoclasts. Stimulation of WT cells by M-CSF and RANKL resulted in a significant 5.8-fold increase in nuclear NF-κB signal versus stimulation by M-CSF alone (n = 4): *P < .005. There was an insignificant increase in nuclear NF-κB signal after stimulation of Sbds-null cells with M-CSF and RANKL: P = .065. (C) Specificity of the NF-κB ELISA was verified with the addition of WT or mutant oligonucleotide competitors. WT oligo, representing the native consensus binding sequence of NF-κB (5′-GGGACTTTCC-3′), significantly inhibited NF-κB binding in the positive control (Jurkat cells stimulated with PMA and A23187), WT and Sbds-null nuclear extracts, whereas the mutant consensus oligonucleotide had no effect. Specificity of 1 signifies 100% specificity (n = 4): *P < .01. (D) Translocation of NFATc1 into the nucleus was evaluated as a second marker of the RANK signaling pathway by performing an ELISA on NFATc1 content in day 4 nuclear extracts. Stimulation of WT cells by M-CSF and RANKL resulted in a significant 2.5-fold increase in nuclear NFATc1 signal versus stimulation by M-CSF alone. There was no increase in nuclear NFATc1 signal after stimulation of Sbds-null cells with M-CSF and RANKL (n = 4): *P < .005. (E) NFATc1 ELISA showed good specificity as the WT oligo, representing the native consensus binding sequence of NFATc1 (5′-AGGAAA-3′), significantly inhibited NFATc1 binding in the positive control (leukoagglutinin-stimulated Jurkat cells), WT and Sbds-null nuclear extracts, whereas the mutant consensus oligonucleotide had no effect (n = 4): *P < .005; **P < .01.

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