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. 2020 Nov 21;2(1):100015. doi: 10.1016/j.xhgg.2020.100015

Figure 4.

Figure 4

Variants p.Glu247Gly and p.Thr244Lys have reduced affinity for 14-3-3β

(A) HEK293 cells were transfected with expression plasmids for wild-type HDAC4, or the p.Thr244Lys or p.Glu247Gly variants as shown, then extracts subjected to immunoprecipitation (IP) with anti-FLAG antibody. Upper panels show western blots of cell extracts using either anti-FLAG (to detect HDAC4) or anti-HA (to detect 14-3-3β); lower panels show western blots of IP samples using the same antibodies, showing both short and long exposures of the anti-HA blot. Data shown for variant p.Glu247Gly are representative of five independent experiments; the p.Thr244Lys variant was tested in a single experiment, as shown here.

(B) Relative binding of 14-3-3β by the p.Glu247Gly variant was quantified from five independent experiments; error bars show standard deviation from the mean value; statistical significance was calculated using Student’s t test, ***p < 0.001; data for p.Thr244Lys were derived from the single experiment shown in (A).

(C) As in (A), showing data from an independent experiment after transfection of expression plasmids for wild-type HDAC4 or the p.Glu247Gly variant only; extended regions of the anti-HA blots are shown to demonstrate C-terminal-truncated products of HDAC4 fragmentation, indicated by asterisks (*).