Fig. 5: B4GalT5 knockdown reduces BMPRIA glycosylation, increases its stability and enhances its intracellular distribution. | Cell Death & Disease

Fig. 5: B4GalT5 knockdown reduces BMPRIA glycosylation, increases its stability and enhances its intracellular distribution.

From: Downregulation of β1,4-galactosyltransferase 5 improves insulin resistance by promoting adipocyte commitment and reducing inflammation

Fig. 5

a On day 6 post-induction, cells were stained with oil red O to assess adipogenesis. b Western blotting of PPARγ, C/EBPα, and 422/aP2 on day 6 post-induction. c RCA-I pulldown assay to analyze BMPRIA and noggin glycosylation level in C3H10T1/2 cells with different treatment. Results were quantified; the fold change describes the change in the proportion of bound divided by total for each protein. d C3H10T1/2 cells treated with NC or B4GalT5 siRNA were then transfected with wild-type Flag-BMPRIA plasmids. Forty-eight hours later, coimmunoprecipitation assays were performed with anti-Flag antibody, followed by lectin blotting assay to detect the RCA-I-binding level. Results were quantified; the fold change describes the change in the proportion of bound RCA-I level divided by the pull-downed Flag. e C3H10T1/2 cells treated with B4GalT5 siRNA or negative control were incubated with cycloheximide (40 μM) for the indicated times on day 0. After treatment, cell lysates were checked by western blotting with anti-BMPRIA antibody. f The BMPRIA protein level after cycloheximide treatment in panel e was quantified, normalization to 0 h, and plotted as shown. g C3H10T1/2 cells with different treatment were performed immunofluorescence staining to demonstrate BMPRIA distribution in the cellular level (Scale bar: 50 μm). h BMPRIA cell surface expression was displayed by flow cytometric analysis. i C3H10T1/2 cells treated with NC or B4GalT5 siRNA were then transfected with wild-type Flag-BMPRIA or 3Q mutant Flag-BMPRIA plasmids. Forty-eight hours later, coimmunoprecipitation assays were performed with anti-Flag antibodies, followed by lectin blotting assay to detect the RCA-I-binding level. Results were quantified; the fold change describes the change in the proportion of bound RCA-I level divided by the pull-downed Flag. j C3H10T1/2 cells were transfected with wild-type Flag-BMPRIA or 3Q mutant Flag-BMPRIA plasmids, 48 h later, cells were incubated with CHX for the indicated times. Cell lysates were monitored by western blotting with anti-Flag antibody. k The Flag levels in Fig. 5J were quantified, normalization to 0 h and plotted as shown. Each data is representative of at least three independent experiments with identical results. *P < 0.05, **P < 0.01, ***P < 0.001

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