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. 2018 Oct 19;27(3):466–474. doi: 10.1038/s41431-018-0282-4

Fig. 3.

Fig. 3

Missense variant in the TBL1Y gene decreases protein stability. a Western blot showing the amount of TBL1Y wild-type (TBL1Y-WT) and mutant (TBL1Y-Asp69Val) proteins in HEK 293 transfected cells, 24 and 48 h after transfection. The results showed a minimum amount of the mutant protein 48 h post transfection. b qRT-PCR; mRNA quantification after 24 and 48 h from transfection. The qRT-PCR results show no differences between wild-type and mutated mRNA expression. c Immunodetection of TBL1Y-WT and TBL1Y-Asp69Val in HeLa-transfected cells. The wild-type and mutated proteins show the same cellular localization, although with different levels of expression. Myc (green), beta tubulin (red), and DAPI (blue). Scale bar: 50 μm. d Western blot experiment showing the amounts of TBL1Y in lysates from TBL1Y-WT or TBL1Y-Asp69Val-transfected HEK 293 cells, collected at the indicated time points after addition of cycloheximide (CHX). Results, quantified densitometrically, are shown in the graphs below the Western blots, where the amount of TBL1Y protein upon treatment of MG132 for 4 h was set as 100%. The genetic variant decreases protein stability and triggers its premature degradation through the proteasome. e Western blot showing the amount of ubiquitinated TBL1Y-WT and TBL1Y-Asp69Val in lysates of MG132-treated or untreated HEK 293 cells, co-transfected with HA-tagged ubiquitin (HA-Ubi). Ubiquitinated TBL1Y proteins were detected after immunoprecipitation with a Myc-specific antibody followed by Western blotting with a HA-specific antibody. f TBL1Y-WT and TBL1Y-Asp69Val immune-precipitations were in vitro digested with trypsin at different time points (15, 30, 180, and 600 min) and then analyzed by LC–MS/MS. Peptides were identified using the X!Tandem search engine. The abundance of tryptic fragments was determined by the log of the intensity of all the peaks obtained from TBL1Y MS/MS spectra. Results are representative of three independent experiments (four replicates/independent experiment) at the indicated time points; shown are mean ± s.e.m.; *P < 0.05; **P < 0.001. The data were analyzed with one-way ANOVA and Bonferroni’s multiple comparison test