Fig. 3: GSK3β interacts with N1-ICD in CLL cells and is involved in N1-ICD ubiquitination.

A N1-ICD was immunoprecipitated (IP) from whole-cell extracts of CLL cells, and the IP lysates were analyzed by Western blot with the anti-NOTCH1 (Val1744) to confirm IP of N1-ICD, and with the anti-GSK3β antibody to detect GSK3β/N1-ICD interaction (n = 3). One representative CLL is shown. B Confocal microscopy images of subcellular localization of GSK3β in a representative CLL sample. CLL cells (n = 3) were stained with the anti-GSK3β antibody (red) and with DAPI for nuclei (blue) and then analyzed by confocal microscopy, with a 63x oil immersion and 1.4 NA objective; scale bar, 10 μm. C, D PLA was performed by using rabbit anti-NOTCH1 (Val1744) and mouse anti-GSK3β antibodies to detect GSK3β/N1-ICD interactions in CLL cells cultured for 1.5 h with 5 µM SB216763 or DMSO (C; n = 3), and by using rabbit anti-NOTCH1 (Val1744) and mouse anti-ubiquitin antibodies to detect N1-ICD/Ubiquitin interactions in CLL cells cultured with 5 µM SB216763 or DMSO for 1.5 h, and with 10 µM MG132 for additional 4 h (D; n = 3). Nuclei were stained with DAPI. In the confocal microscopy images, red spots indicate GSK3β/N1-ICD (C) and N1-ICD/Ubiquitin (D) interactions. Images were acquired by using confocal microscopy with a 63x oil immersion and 1.4 NA objective; scale bar, 10 μm. One representative CLL is shown. In the bottom panel (C) and in the right panel (D), bar graphs ± SEM show quantitative analysis of the PLA signals of three samples. ****P < 0.0001; **P < 0.01; *P < 0.05 according to unpaired Student’s t-test.