HeLa Flp-in Trex cells stably expressing Flag-empty, Flag-PINK1 WT (wild-type) or Flag-PINK1 KI (Kinase inactive) were transfected with HA-Rab1B for 24h before Doxycycline (0.1μg/ml) treatment for 24h. All cells were treated with 10μM CCCP for 3h. Whole cell lysates (0.25 mg) were immunoprecipiated with anti-HA agarose and immunoblotted with Rab1A/1B phospho-Ser114/Ser111 antibodies. Binding of the primary antibody was detected using anti-sheep (1:10,000) and visualised with ECL. Note:5th bleed works the best for detecting Rab1B Ser111 phosphorylation. Only Rab1B were test because HA-Rab1A was not expressed well by transient transfection.