The Effect of the NFκB-USP9X-Cx43 Axis on the Dynamic Balance of Bone Formation/Degradation during Ossification of the Posterior Longitudinal Ligament of the Cervical Spine

Figure 1

Cx43 interacted with USP9X and USP9X inhibited Cx43 ubiquitination. (a) Cx43 expression level from ligament fibroblast cells stably transfected with Cx43-Flag or vector alone was detected by Western blot using anti-Flag or anti-Cx43 antibodies. (b) Proteins precipitated using anti-Flag antibodies were resolved by SDS-PAGE and stained using Coomassie Brilliant Blue. (c) Western blots of proteins precipitated using anti-USP9X antibodies and anti-Cx43 antibodies. (d) Western blots showing that USP9X was successfully knocked down at the protein level. USP9X KD caused Cx43 downregulation. (e) qPCR results showing that USP9X was successfully knocked down at the mRNA level, and no effect was observed on Cx43 expression. (f) Western blots showing that USP9X was successfully overexpressed at the protein level, and USP9X overexpression caused Cx43 upregulation. (g) qPCR results showing that USP9X was successfully overexpressed at the mRNA level, and no effect was observed on Cx43 expression. (h) Western blots showing that primary ligament fibroblast treatment silenced USP9X with MG132 (10 μM) for 4 h significantly upregulating Cx43 expression. (i) Effects of USP9X silencing on Cx43 ubiquitination. (j) Effects of USP9X overexpression on Cx43 ubiquitination. , vs. shNC. ^## vs vector. Ns: not significant.

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