FIG. 6.

GSK-3β is released from an axin-bound complex following trophic deprivation. (A) To determine whether GSK-3β changed interaction partners in response to stress, cerebellar granule neurons (7 DIV) were deprived of trophic support for 2 h, and protein complexes were separated by gel filtration. Fractions (1.5 ml each) (fractions 1 to 55) were collected after the void volume (20 ml) was eluted. Fractions 9 to 49 (7.5%) were loaded alongside equal proportion (7.5%) of total input (Input) and insoluble pellet (Pellet) and immunoblotted as shown. P-GSK, phosphorylated GSK. (B) GSK-3β intensity was quantified from nonsaturated exposures and expressed as a percentage of the total expression. Following trophic deprivation, there is a reduction in the amount of HMW GSK-3β and an increase in the LMW GSK-3β (∼50 kDa). MM, molecular mass markers. (C) Lysates from HEK-293 cells expressing Venus-axin-GID were used to characterize antibodies generated against axin. Antiaxin antibodies were used to immunoprecipitate Venus-axin (IP: Axin). Whole-cell lysate is shown in the right panel. (D) To determine whether GSK-3β-axin interaction was altered in response to stress, 7 DIV cerebellar granule neurons were deprived of trophic support for 4 h. Axin-bound GSK-3β was isolated by immunoprecipitating endogenous axin. A representative exposure is shown. Quantified data from three replicates (means plus standard errors of the means [error bars]) show that there is a significant decrease in axin-bound GSK-3β following trophic deprivation (*, P < 0.05). α-Axin, antiaxin antibody.