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. 2012 Dec 13;3(12):e444. doi: 10.1038/cddis.2012.184

Figure 5.

Figure 5

AUL12 induces an interaction between phosphorylated GSK-3α/β and Bax, which translocates onto mitochondria and contributes to PTP induction and cell death. (a) On the left, GSK-3α/β immunoprecipitation in SAOS cells treated or not for 2 h with 40 μM AUL12; where indicated, cells were pre-incubated with IND ( 2 μM, 3 h) or NAC (1 mM, 1 h). The Western immunoblot was probed with Bax, whereas β-catenin is used as a positive control of co-immunoprecipitation with GSK-3α/β in starved cells (STV); NC, negative control. On the right, p(Y216)GSK-3α/β immunoprecipitation in SAOS cells treated or not for 2 h with 40 μM AUL12. The Western immunoblot was probed with GSK-3α/β and Bax. In both panels, IgG is used as a loading control. (b) Bax immunoprecipitation in SAOS cells treated or not for 2 h with 40 μM AUL12; where indicated, cells were pre-incubated with IND, as in (a). The Western immunoblot was probed with GSK-3α/β with Bax, and with IgG as a loading control; NC, negative control. (c) Western immunoblot on cytosolic and mitochondrial fractions of SAOS-2 cells treated or not for 2 h with 40 μM AUL12; where indicated, cells were pre-incubated with IND, as in (a). To check for fraction purity, the blot was also probed with β-catenin as a cytosolic marker and with TOM20 as a mitochondrial marker. (d) CRC experiments on whole cells treated with the reported concentrations of AUL12 (with or without a 1 h of preincubation with a Bax inhibiting peptide (BIP, 60 μM). Mitochondrial Ca2+ uptake in the various conditions is compared with that measured in unstimulated cells. (e) Death induction on SAOS-2 cells exposed to different concentrations of AUL12 with or without pre-incubation with the BIP (60 μM) is analyzed by cytofluorimetry with a propidium iodide/Annexin V-FITC double staining, as in Figure 1b. Viable cells reported in the bar graph are negative for both fluorophores. All along the figure, bar graphs report mean±S.D. values (n=3). Statistical significance was measured with a Student's t test and is indicated by asterisks (*P<0.01, **P<0.005)