Mouse anti rab4

Treated cells were collected in Laemmli sample buffer, sonicated and boiled. Samples were run on NuPage 4%–12% Bis-Tris gel (Life Technologies), transferred to PVDF membranes (Perkin Elmer), blocked in 5% skimmed milk and incubated successively with primary and secondary-HRP coupled antibodies, and finally visualized with ECL (Thermo Scientific) or Luminata Crescendo (Millipore) HRP reagents depending on the strength of the signals. Signals were captured on Amersham Hyperfilm ECL (GE Healthcare), developed using a Xograph Compact X4 film developer and analyzed using ImageJ software (National Institutes of Health, USA). Signals used for quantifications were captured at a pre-saturation intensity. Results are derived from triplicate biological repeats and represent signals that were normalized to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control and to the signals from resting cells. Antibodies used were mouse anti-Rab4 (BD Biosciences 610888), mouse anti-Rab5 (BD Biosciences 610282), rabbit anti-Rab7 (Cell Signaling Technologies D95F2), rabbit anti-Rab8 (Cell Signaling Technologies D22D8), rabbit anti-Rab11 (Life Technologies, 715300), mouse anti-GAPDH (Santa Cruz 0411), and rabbit anti-GAPDH (Cell Signaling Technologies 14C10).

Rabbit anti-Xenopus laevis ESCRT-II polyclonal antibody was made in the Blower laboratory; other antibodies were obtained as follows: mouse anti-acetylated α-tubulin (Sigma cat. no. T6793), mouse anti-α-tubulin (Sigma cat. no. T6074), mouse anti-Rab4 (BD Biosciences cat. no. 610888), mouse anti-Rab5 (BD Biosciences cat. no. 610281), mouse anti-Rab7 (Sigma cat. no. R8779), mouse anti-Rab11 (BD Biosciences cat. no. 610656), rabbit anti-β-tubulin (Abcam cat. no. ab6046), mouse anti-DCC intracellular domain (BD Biosciences cat. no. 554223), goat anti-DCC extracellular domain (R&D Systems cat. no. AF844), rabbit anti-RPL5 (Proteintech Europe cat. no. 15430-1-AP).