Figure 2. Bax and Bcl2 are associating partners of Diva.

a Co-immunoprecipitation (Co-IP) and reverse immunoprecipitation of Diva, Bax and Bcl2 after 30 nM of H₂O₂ treatment. Total protein was isolated from treated PC12 cells after 0 hrs, 2 hrs, 4 hrs and 8 hrs of H₂O₂ treatment. Co-IP was first conducted with Diva antibodies to determine how Bax and Bcl2 interact with Diva during apoptosis. Reverse Co-IP was later conducted using Bax and Bcl2 antibodies to confirm the changes in protein binding. B-actin was used as an input control to ensure equal loading of total protein. Rabbit IG was used as a negative control to ensure no unspecific binding of IGs to antibodies occurred. Co-IP of Diva to B-actin was performed and used as a negative control (no bands shown). Pre-clearing of agarose beads was performed to ensure no unspecific binding of antibodies to beads occurred (no bands shown). b Duolink of Diva and Bax at 0 hr and 4 hr after H₂O₂ treatment to show the degree of interaction between Diva and Bax during apoptosis. c Duolink of Diva and Bcl2 at 0 hr and 8 hr after H₂O₂ treatment to show the degree of interaction between Diva and Bcl2 during apoptosis. Red Cy3 stained for interaction between two proteins while blue DAPI stained for the nucleus. Bar  = 20 µm.