Rab8b

HA-Rab8b was transiently transfected into Flp In HEK293 stable cell lines expressing PINK1-Flag tagged Wildtype (WT). Cells were induced for PINK1 expression with doxycyclin for 24 hrs and stimulated with and without CCCP (mitochondrial depolarizing agent) for 3 hours. HA-Rab8b S111A mutant was transfected into PINK1-Flag WT stable cell lines. Cells were induced for PINK1 expression with doxycyclin for 24 hours and stimulated with CCCP (mitochondrial depolarizing agent) for 3 hours. 1 mg of whole cell lysate was immunoprecipitated for Rab8b using 10 ul of HA- agarose affinity resin. Proteins were resolved by SDS-PAGE and subjected to western blot analysis. The blot was probed with the bleeds of anti-Rab8b phospho Ser 111 S504D antibody as indicated. Antibody recognises HA-Rab8b when it is expressed in cells stably expressing PINK1 under conditions of mitochondrial depolarization and not under basal conditions. Also the specificity of the antibody is confirmed with loss of recognition of HA-Rab8b S111A mu