Journal: The Journal of Biological Chemistry
Article Title: Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans -Golgi Network *
doi: 10.1074/jbc.M110.137414
Figure Lengend Snippet: Endogenous CAPS1 is associated with the Golgi membrane. A and B, electron micrograms of submicrosomal fractions affinity-purified using immune magnetic beads. Cerebellar microsomal fractions of P21 mice were incubated with magnetic beads coated with anti-CAPS1 antibody (A) or anti-VAMP2 antibody (B), and immunoaffinity-purified submicrosomal fractions were subjected to electron microscopic observation. Scale bars, 200 nm. C, Western blot analysis of submicrosomal fractions affinity-purified using normal rabbit IgG, anti-CAPS1 antibody, or anti-VAMP2 antibody immunomagnetic beads. Each immunopurified fraction was immunoblotted with anti-CAPS1, anti-VAMP2, anti-VAMP4, anti-SNAP25, anti-Bcl-2, anti-BiP/GRP78, anti-pan-ARF (1D9), anti-GM130, anti-GS28, anti-p115, anti-Stx5, anti-Stx6, anti-Stx16, and anti-Vti1a antibodies. Anti-pan-ARF (1D9) antibody recognizes all of the ARF family proteins (supplemental Fig. S3).
Article Snippet: The following primary antibodies were also used for Western blotting: rabbit polyclonal anti-CAPS2 (1:1,000 dilution) ( 5 ), mouse monoclonal anti-FLAG (1:1,000 dilution; catalog no. F1804, Sigma), rat monoclonal anti-hemagglutinin epitope (HA) (1:1,000 dilution; catalog no. 1867423, Roche Applied Science), mouse monoclonal anti-VAMP2 (1:10,000 dilution; catalog no. 104211, Synaptic Systems), rabbit polyclonal anti-VAMP4 (1:1,000 dilution; catalog no. PA1-768, Affinity BioReagents), mouse monoclonal SNAP25 (1:2,500 dilution; catalog no. SMI-81, Sternberger), mouse monoclonal anti-ARF (clone 1D9) (1:500 dilution; catalog no. ab2806, Abcam), mouse monoclonal anti-GM130 (1:250 dilution; catalog no. 610822, BD Biosciences), mouse monoclonal anti-GS28 (1:1,000 dilution; catalog no. 611184, BD Biosciences), mouse monoclonal anti-p115 (1:1,000 dilution; catalog no. 612260, BD Biosciences), mouse monoclonal anti-Bcl-2 (1:500 dilution; catalog no. 610538, BD Biosciences), mouse monoclonal anti-BiP/GRP78 (1:250 dilution; catalog no. 610978, BD Biosciences), rabbit polyclonal anti-syntaxin 5 (1:1,000 dilution; catalog no. 110053, Synaptic Systems), mouse monoclonal anti-syntaxin 6 (1:2,500 dilution; catalog no. 610635, BD Biosciences), rabbit polyclonal anti-syntaxin 16 (1:1,000 dilution; catalog no. 110162, Synaptic Systems), mouse monoclonal anti-Vti1a (1:2,500 dilution; catalog no. 611220, BD Biosciences), and goat polyclonal anti-GST (1:1,000 dilution; catalog no. 27-4577, Amersham Biosciences).
Techniques: Affinity Purification, Magnetic Beads, Incubation, Purification, Western Blot
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