Rabbit gm130 antibody

Sugars, nucleotides, mouse anti-FLAG antibody and FLAG affinity beads (anti-FLAG M2 affinity gel) were purchased from Sigma. EnzCheck pyrophosphate assay kit was obtained from Invitrogen. PNGaseF enzyme was from NEB. Rabbit GM130 antibody was from Abcam. Drosophila strains with GAL4 drivers were obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN). DmCSAS mutants and the FLAG-tagged DmCSAS construct were previously described [8 (link)]. GFP-tagged human CMP-sialic acid synthetase construct was a gift from Michael Betenbaugh (The Johns Hopkins University).

The CSAS-DA mutant protein was expressed in Drosophila larval neurons essentially as described previously [8 (link)]. Briefly, the expression was induced in the brain using UAS-GAL4 system [12 (link)]. Third instar larval brains were dissected in ice-cold Ringers solution, rinsed and fixed in fresh Fixative solution (4% paraformaldehyde, 50 mM NaCl, 0.1 M Pipes pH 7.2) for 20 minutes at room temperature on nutator. Fixed tissue was washed with PBT (PBS pH 7.5, 0.1% Triton X100, 1% BSA, 0.01% sodium azide) and analyzed by immunostaining and epifluorescent microscopy. CSAS-DA-FLAG was detected using mouse anti-FLAG M2 (Sigma) primary antibody (1:2,000). Rabbit GM130 antibody (Abcam) was used at 1:100 dilution. Incubation with fluorescent secondary antibodies was carried out essentially as described previously [15 (link)]. The following secondary antibodies and corresponding dilutions were used: goat anti-mouse Alexa-546 and goat anti-rabbit Alexa-488, 1:250 (Molecular Probes). Immunostained brains were mounted in Vectashield medium (Vector Laboratories) and analyzed using Zeiss Axioplan 2 microscope with ApoTome module for optical sectioning.