Figure 2. ACSS2 phosphorylation at S659 exposes the NLS of ACSS2 to bind to importin α5.

(C–H) Immunoblotting analyses were performed with the indicated antibodies.

(A) Schematic of ACSS2 showing its potential NLS predicted by the NLStradamus tool.

(B and C) U87 cells expressing the indicated Flag-ACSS2 proteins were deprived of glucose for 1 h. Immunofluorescent analyses were performed with an anti-Flag antibody and the percentage of nuclear ACSS in 20 cells in each group were quantitated (right panel) using the ImageJ software program (B). Total cell lysates and cytosolic and nuclear fractions were prepared (C). A two tailed Student’s t test was used. ∗ represents P < 0.001. (D) U87 cells expressing the indicated SFB-tagged importin α proteins were deprived of glucose for 10 min. A pull-down assay with streptavidin agarose beads was performed.

(E) U87 cells were deprived of glucose for 10 min. Immunoprecipitation with an anti-importin α5 antibody was performed.

(F) U87 cells with or without importin α5 depletion were deprived of glucose for 1 h. Total cell lysates and cytosolic and nuclear fractions were prepared.

(G) Purified GST-importin α5 was mixed with the indicated purified His-ACSS2 proteins in the presence or absence of active AMPK. A GST pull-down assay was performed.

(H) U87 cells expressing the indicated Flag-ACSS2 proteins were deprived of glucose for 10 min. Immunoprecipitation with an anti-Flag antibody was performed.

(I) Parental and the indicated U87 cells with knock-in of ACSS2 S659A or R664/665A were deprived of glucose for 1 h. Immunofluorescent analyses were performed with an anti-ACSS2 antibody. The percentage of nuclear ACSS in 20 cells in each group were quantitated (right panel) using the ImageJ software program. A two tailed Student’s t test was used. ∗ represents P < 0.001.

See also Figure S2.