Biii tubulin

Cells were washed in PBS and fixed for 10 minutes in 4% paraformaldehyde followed by 30 minutes blocking and permeabilization in 5% normal goat serum with 0.2% Triton-X 100. For Golgi morphology, cells were incubated over night at 4°C with anti GM130 (BD bioscience, 1:500) and alpha-tubulin (Sigma, 1:4000). Cells were washed three times and incubated in secondary antibody (Alexa-488 and Alexa 594 1:1000) for 1 hr at room temperature. Cells were mounted in Prolong Gold with DAPI to visualize nuclei. Golgi morphology was classified as normal, intermediate, diffuse or absent by blinded observers with at least 50 cells from three separate experiments, and statistical significance was determined by Chi² analysis. For detection of SMN in COPA depleted NSC-34 cells, cells were plated on 18mm glass coverslips coated with PDL and cultured in DMEM/F12 with 1% FBS for 72 hours to induce neurite outgrowth. Cultures were then infected with 4×10⁶ lentivirus particles for 48 hours. Alpha-COP knockdown was confirmed by Western Blot analysis. Cells were stained with BIII-tubulin (rabbit, 1:1000 Cell Signaling Technologies) to visualize neurites and growth cones, and Mansma2 (1:50) to visualize SMN. For cells transfected with YFP-KDELR-VSVGts045 cells were immunolabeled with alpha-tubulin (Sigma, 1:5000) and Alexa 594 (goat anti-mouse 1:1000).