Figure 2.
Figure 2. GSK3 is recruited to DP to modulate DP–IF complexes. (A) Endogenous DP associates with HA-GSK3 in HA immunoprecipitations in HEK 293 cells, assessed by immunoblotting. (B) SCC9 cells were fixed, and DP and GSK3 associations were analyzed by PLA analysis using anti-DP (NW6) and anti-GSK3 antibodies. Direct interactions (red) and nuclei (DAPI, blue). Bars, 10 µm. (B′) PLA fluorescent spots are counted and divided by the total number of nuclei in an image. Data were obtained from antibody pairs for >500 cells per experiment for three independent experiments. *, P < 0.001. Error bars indicate SEM. (C and C′) Phospho-DP in LiCl- or NaCl-treated SCC9s, assessed by immunoblotting. Full-length endogenous DP has two known isoforms, DPI (310 kD) and DPII (250 kD) that are generated by alternative splicing. Both of these contain S2849 and are recognized by the phospho-DP antibody. Densitometry quantification of three independent experiments. Error bars indicate SEM. (D) DP phosphorylation in siCtr or siGSK3-treated cells, assessed by immunoblotting. (E and E′) Phospho-DP in control or caGSK3 transfected cells, and lysates were assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (F) S-tag affinity pull-down of HA-GSK3 and DP S-tag (DP CT) HEK 293 cells, assessed by immunoblotting. (G) NaCl-, LiCl-, or Ly-294,002 (GSK3 activator)–treated DP S-tag (DP CT) expressing cell lysates, assessed by immunoblotting. DP S-tag (DP CT) is present as a doublet where the lower band (32 kD) contains phosphorylation of S2849, whereas the higher band (red arrow, 36 kD) contains both phosphorylation of S2849 and downstream GSK3-phosphorylated sites (see cartoon in Fig. 3 A). (G′) Densitometry quantification of G from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) NaCl- or LiCl-treated DP S-tag (DP CT) cells were transfected with control or caGSK3, assessed by immunoblotting. Long exposure and short exposure of DP CT blot shown to visualize upper phospho-states of DP (36 kD). (H′) Densitometry quantification of phospho-DP divided by total DP from three independent experiments. Error bars indicate SEM.

GSK3 is recruited to DP to modulate DP–IF complexes. (A) Endogenous DP associates with HA-GSK3 in HA immunoprecipitations in HEK 293 cells, assessed by immunoblotting. (B) SCC9 cells were fixed, and DP and GSK3 associations were analyzed by PLA analysis using anti-DP (NW6) and anti-GSK3 antibodies. Direct interactions (red) and nuclei (DAPI, blue). Bars, 10 µm. (B′) PLA fluorescent spots are counted and divided by the total number of nuclei in an image. Data were obtained from antibody pairs for >500 cells per experiment for three independent experiments. *, P < 0.001. Error bars indicate SEM. (C and C′) Phospho-DP in LiCl- or NaCl-treated SCC9s, assessed by immunoblotting. Full-length endogenous DP has two known isoforms, DPI (310 kD) and DPII (250 kD) that are generated by alternative splicing. Both of these contain S2849 and are recognized by the phospho-DP antibody. Densitometry quantification of three independent experiments. Error bars indicate SEM. (D) DP phosphorylation in siCtr or siGSK3-treated cells, assessed by immunoblotting. (E and E′) Phospho-DP in control or caGSK3 transfected cells, and lysates were assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (F) S-tag affinity pull-down of HA-GSK3 and DP S-tag (DP CT) HEK 293 cells, assessed by immunoblotting. (G) NaCl-, LiCl-, or Ly-294,002 (GSK3 activator)–treated DP S-tag (DP CT) expressing cell lysates, assessed by immunoblotting. DP S-tag (DP CT) is present as a doublet where the lower band (32 kD) contains phosphorylation of S2849, whereas the higher band (red arrow, 36 kD) contains both phosphorylation of S2849 and downstream GSK3-phosphorylated sites (see cartoon in Fig. 3 A). (G′) Densitometry quantification of G from three independent experiments. *, P < 0.001. Error bars indicate SEM. (H) NaCl- or LiCl-treated DP S-tag (DP CT) cells were transfected with control or caGSK3, assessed by immunoblotting. Long exposure and short exposure of DP CT blot shown to visualize upper phospho-states of DP (36 kD). (H′) Densitometry quantification of phospho-DP divided by total DP from three independent experiments. Error bars indicate SEM.

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