Fig. 3: USP49 interacts with PAX9 and MSX1 to regulate their protein turnover. | Cell Death & Differentiation

Fig. 3: USP49 interacts with PAX9 and MSX1 to regulate their protein turnover.

From: Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis

Fig. 3

A The interactions between endogenous USP49 and PAX9 proteins were examined in hDPSCs. Cell lysates from hDPSCs were immunoprecipitated and immunoblotted with specific USP49 or PAX9 antibodies. The presented immunoblots are representative of three independent experiments (n = 3). B The interactions between endogenous USP49 and MSX1 proteins were examined in hDPSCs. Cell lysates from hDPSCs were immunoprecipitated and immunoblotted with specific USP49 or MSX1 antibodies. The presented immunoblots are representative of three independent experiments (n = 3). C Myc-PAX9 and HA-USP49 were co-transfected into HEK293 cells for immunoprecipitation. The presented immunoblots are representative of two independent experiments (n = 2). D Flag-MSX1 and GFP-USP49 were co-transfected into HEK293 cells. Samples were immunoprecipitated using their respective antibodies and immunoblotted using the indicated antibodies. GAPDH was used as the loading control. The presented immunoblots are representative of two independent experiments (n = 2). E hDPSCs were subjected to the Duo-link PLA assay to check the interaction between USP49 and PAX9; USP49 and MSX1 using their specific antibodies. Scale bar: 25 μm. The in situ USP49-PAX9 or USP49-MSX1 interaction (PLA dots) was observed when USP49 and PAX9 or MSX1 were immunostained together, but not when they were stained with individual antibodies. The presented microscopic images are representative of three independent experiments (n = 3). F Immunostaining with endogenous USP49 and PAX9 or MSX1 antibodies was performed in hDPSCs. As the specific antibodies against USP49, PAX9 or MSX1 were derived from the same species, we transduced hDPSCs with Flag-USP49 and performed staining with Flag- and PAX9- or MSX1-specific antibodies. DAPI was used for nuclear staining. Scale bar: 100 µm. The presented microscopic images are representative of three independent experiments (n = 3). G The half-life of PAX9 was determined in the Mock, USP49 KO, and USP49 KO cells reconstituted with USP49 after treatment with CHX for the indicated time points. Densitometric analysis of PAX9 expression (normalized to GAPDH) is reported under the blot and represents the mean of three independent experiments (n = 3). H The half-life of MSX1 was determined in the Mock, USP49 KO, and USP49 KO cells reconstituted with USP49 after treatment with CHX for the indicated time points. Densitometric analysis of MSX1 expression (normalized to GAPDH) is reported under the blot and represents the mean of three independent experiments (n = 3). I The half-life of PAX9 was determined in the Mock, USP49 KO, and USP49 KO cells reconstituted with USP49CA after treatment with CHX for the indicated time points. Densitometric analysis of PAX9 expression (normalized to GAPDH) is reported under the blot and represents the mean of three independent experiments (n = 3). J The half-life of MSX1 was determined in the Mock, USP49 KO, and USP49 KO cells reconstituted with USP49CA after treatment with CHX for the indicated time points. Protein band intensities were estimated using the ImageJ software with reference to the GAPDH control. The band intensity for PAX9/GAPDH or MSX1/GAPDH was represented below the blots. Densitometric analysis of MSX1 expression (normalized to GAPDH) is reported under the blot and represents the mean of three independent experiments (n = 3).

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