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. 2016 Aug 2;99(2):489–500. doi: 10.1016/j.ajhg.2016.06.022

Figure 3.

Figure 3

Impact of the DNAJB13 p.Met278Arg Substitution on Protein Amounts, Protein Stability, and Proteasomal Degradation

(A) Left: CHO-K1 cells were transiently transfected with 0.5 μg of the plasmids encoding FLAG-DNAJB13_WT and FLAG-DNAJB13_Met278Arg. After 24 hr, the proteins were extracted, and equal amounts of protein extracts were subjected to SDS-PAGE on a 14% gel and immunoblotted with an anti-FLAG antibody. β-tubulin was used as a loading control. Right: the FLAG signal was quantified with ImageJ software and normalized to the amount of β-tubulin. The results are shown as the mean of ten independent experiments and are expressed as a percentage of the WT condition. Asterisks indicate that the mean is significantly different; t test p value = 0.0001.

(B) Left: CHO-K1 cells were transiently transfected with 0.5 μg of the plasmids encoding FLAG-DNAJB13_WT and FLAG-DNAJB13_Met278Arg. Cells were treated with 200 μg/mL of cycloheximide (CHX), and protein extracts were prepared at the indicated time points. Equal amounts of protein extracts were subjected to SDS-PAGE and immunoblotted with an anti-FLAG antibody. β-tubulin was used as a loading control. Right: the FLAG signal was quantified with ImageJ software and normalized to the amount of β-tubulin. The results are shown as the mean of three independent experiments and are expressed as a percentage of the WT, non-treated condition (p value = 0.008). Obtained values were plotted against time, and the half-lives of the DNAJB13 proteins were quantified.

(C) Left: CHO-K1 cells were transiently transfected with 0.5 μg of the plasmids encoding FLAG-DNAJB13_WT and FLAG-DNAJB13_Met278Arg. After 24 hr, the cells were treated with 20 μM of MG132 for 6 hr and then lysed for protein extraction in Laemmli sample buffer. Samples were subjected to SDS-PAGE on 14% gels and immunoblotted with an anti-FLAG antibody. β-tubulin was used as a loading control. Right: the FLAG signal was quantified with ImageJ software and normalized to the amount of β-tubulin. The results are shown as the mean of four independent experiments and are expressed as a percentage of the WT, non-treated condition. Asterisks indicate that the means are significantly different; t test p values = 0.04 (a) and 0.05 (b). “ns” indicates that the mean is not significantly different; t test p value = 0.25 (c).