Rab1a

Whole-cell lysates were harvested using RIPA buffer (Cell Signaling Technology) containing protease inhibitor cocktail, the PhosSTOP phosphatase inhibitors and PMSF (Roche, Basel, Switzerland). 30 μg of lysate protein were then fractionated in SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, USA). After blocking antigen, the membranes were incubated with primary antibodies and the corresponding secondary antibodies. The primary antibody used in this assay included Rab1A (Santa Cruz, USA) and GAPDH, S6K1, P-S6K1(T398), E-cadherin, N-cadherin, Vimentin, ZO-1, H2AX, γ-H2AX, Rad50, Mer11, P-NBS1(Ser343), P-ATM(Ser1981), Ku80, DNA-PKcs, β-catenin, GSK-3β, P-GSK-3β(Ser9) (Cell Signaling Technology) and Rad51 (Proteintech Group, USA). Finally, proteins were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Waltham, MA, USA).

Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 5% milk powder in PBS containing 0.1% Triton-X-100 (PBST) for 1 h, RT. Primary antibodies were applied for 1–2 h, RT or overnight, 4 °C, washed in PBST, then incubated with anti-mouse IgG or anti-rabbit IgG conjugated to horseradish peroxidase for 1 h. Following washing in PBST, membranes were visualized either using ECL (GE Healthcare) and exposed to Kodak X-Omat S film or analyzed directly on a LICOR Odyssey imager (LI-COR Biosciences, Lincoln, Ne, USA) using antibodies conjugated to fluorescent markers. Antibodies: GM-130 (EP892Y, Abcam, Cambridge, UK), GAPDH (E1C604-1, Enogene, Calnexin (SPA-860, Stressgen, San Diego, CA, USA), lamin A and C (3262, 3931), Rab1a (Santa Cruz Biotechnology, Dallas, TX, USA), Rab24 (BD bioscience, San Jose, CA, USA), gC (ab6509, Abcam, Cambridge, UK), VAPB mouse monoclonal antibody (66191-1, Proteintech, Manchester, UK), and US3 (rabbit polyclonal) and UL34 (rabbit polyclonal) antibodies were kindly provided by Dr. Thomas Mettenleiter and Dr. Barbara Klupp (Friedrich-Loeffler-Institut, Greifswald, Germany). Dr. Christopher C.J. Miller (Kings College London, London, UK) kindly provided the VAPB rabbit antibody.