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. 2020 May 21;106(6):830–845. doi: 10.1016/j.ajhg.2020.04.015

Figure 5.

Figure 5

Functional Tests of SOX6 Missense Variants

(A) Stability and intracellular distribution of SOX6 variants. COS-1 cells were transfected with plasmids encoding 3FLAG-tagged SOX6 WT and variant proteins, as indicated. Cytoplasmic (C) and nuclear (N) extracts were tested via immunoblotting with a FLAG antibody. As expected, the P84 protein was enriched in nuclear extracts, and β-actin was enriched in cytoplasmic extracts. The distribution of SOX6 relative to β-actin in the nuclear versus cytoplasmic compartment is indicated underneath the blots. The migration of protein markers (Mr in k values) is indicated.

(B) Ability of SOX6 variants to homodimerize. Lysates of COS-1 cells, transfected as described in (A), were incubated without (0) or with 0.001%, 0.03%, or 0.01% glutaraldehyde. SOX6 was visualized via immunoblotting with a FLAG antibody. The migration of protein standards (Mr in k values) and SOX6 monomers (1×) and homodimers (2×) is indicated.

(C) Ability of SOX6 variants to bind DNA. An Acan enhancer probe was used in EMSA with no protein extract (−) or with extracts from COS-1 cells transfected with expression plasmids for no protein (none), SOX6 WT, or variant proteins, as indicated. SOX6-probe complexes (indicated with a black arrowhead) and free probes (indicated with a gray arrowhead) were resolved by electrophoresis.

(D) Ability of SOX6 variants to synergize with SOX9 in transactivation. HEK293 cells were transfected with an Acan reporter, a control reporter, and plasmids encoding no SOX (none), SOX9, SOX6 WT, or SOX6 variant proteins, as indicated. The amounts of SOX plasmids are indicated too. Acan reporter activities were normalized for transfection efficiency. They are presented as the mean ± standard deviation obtained for triplicates in an experiment representative of five independent ones. The arrowhead in the middle panel points to the amount of WT SOX6 plasmid (200 ng) that was also used for variant plasmids in the right panel.

(E) Interference of SOX6 variant proteins with WT SOX6 in transactivation. HEK293 cells were transfected as described in (D) with the indicated types and amounts of plasmids encoding no protein (none) or SOX proteins. Acan reporter activities were normalized for transfection efficiency. They are presented as the mean ± standard deviation obtained for triplicates in an experiment representative of four independent ones.