(A) The indicated littermate wild-type and knock-in or knock-out MEF cells were treated ±100 nM MLi-2 for 90 min and lysed. The cell lysates (10 µg) were immunoblotted with the indicated antibodies and the membranes developed using the Odyssey CLx Western Blot imaging system (all at 1 µg/ml antibody). Similar results were obtained in two independent experiments. (B) Quantitation of immunoblots was undertaken by analysing pRab8/tubulin (Bi) or pRab10/tubulin (Bii). (C) As in (A) except that Rab12 was immunoprecipitated using sheep polyclonal total Rab12 antibody from 5 mg of each of the indicated cell extracts. Ninety percent of the immunoprecipitates was immunoblotted with the phospho-Rab12 pSer106 antibody and 1% immunoblotted with the total Rab12 antibody (all at 1 µg/ml antibody and for the phospho-Rab12 pSer106 antibody, 10 µg/ml of dephospho-peptide was also included). Immunoblot (A) developed with LI-COR, and immunoblot (C) developed with ECL.