Figure 3. RANKL or LiCl enhances GSK3β-hnRNPK interaction.

(a) Co-immunoprecipitations of hnRNPK and GSK3β using an anti-hnRNPK antibody (IP-K) performed with RAW264.7 cells treated with RANKL for the indicated durations, revealed by Western blotting with appropriate antibodies. Same co-IP experiments were also performed using non-immune mouse IgG as negative control (IP-Ctrl). Western blotting reveals the increased amount of GSK3β co-immunoprrecipitated with hnRNPK under RANKL induction. The total amounts of hnRNPK and GSK3β in the whole cell lysate were set as internal control. (b) RAW264.7 cells were treated or not with 10 mM LiCl during 8 hours, then harvested and lysed. Total and Ser9 phosphorylated GSK3β levels were assessed by Western blotting using appropriate antibodies. (c) Immunoprecipitation experiments using anti-GSK3β antibody were carried out with cell protein extracts of the cells treated as described in (b). GSK3β and hnRNPK in the immunoprecipitates were revealed by Western blotting using the corresponding antibodies. Non-immune IgG (IgG) was used as a negative control for the specific anti-GSK3β antibody. The numbers under each protein band in the blots indicate the fold change after normalization using GAPDH and in comparison with the control group. Gels were run under the same experimental conditions. For better clarity and conciseness of the presentation, cropped blots are shown. The raw uncropped images can be found in the Supplementary Figure 1. The cropping lines are indicated by black lines both in cropped and uncropped images. The results shown are representative of at least 3 experiments.