Supplementary Materials Supplemental Data supp_287_13_10178__index. disassembled into its element proteins that migrated at their monomer molecular weight on native PAGE. FABP1, freed from the complex, was now able to bind to intestinal ER and generate the pre-chylomicron transport vesicle (PCTV). No increase in ER binding or PCTV generation was observed in the absence of PKC or ATP. We conclude that phosphorylation of Sar1b disrupts the FABP1-made up of four-membered 75-kDa protein complex in cytosol enabling it to bind to the ER and generate PCTV. of the gel). The migration in the gel of Sar1a and Sar1b is usually shown at the of the gel. Detection was by ECL. Antibody Specificity Immunogenic peptides used to generate antibodies to SVIP and Sar1 and the recombinant protein, FABP1, were incubated with their respective antibodies to determine whether the signal on immunoblot generated by the antibody could be extinguished. In each case, the antibody gave a strong signal for the protein against which it was directed in intestinal cytosol. This signal was completely blocked purchase AZD2171 by prior incubation of the antibody with its immunogenic peptide (Sar1, SVIP) or recombinant protein (FABP1). These data suggest the purchase AZD2171 monospecificity of the antibodies employed. Antibody inhibition was not done for Sec13 because the immunogenic peptide was not available, although the antibody has been characterized previously (19). Preparation of Cytosol and Labeling of Enterocytes Enterocytes from the proximal half of male Sprague-Dawley rat small intestine were isolated and radiolabeled with [3H]oleate as described previously (8). In brief, the isolated enterocytes were incubated with albumin-bound [3H]oleate for 30 min at 35 C and washed twice with PBS formulated with 2% BSA to eliminate the surplus [3H]oleate. The tagged enterocytes were homogenized using a Parr bomb, and the cytosol was isolated. The cytosol was dialyzed against Buffer A (0.25 m sucrose, 30 mm HEPES (pH 7.2), 30 mm KCl, 5 mm MgCl2, 5 mm CaCl2, 2 mm DTT) overnight at 4 C and concentrated 5-fold using a 50-ml Amicon filter with a YM10 membrane (Amicon, Beverly, MA). This cytosol was further concentrated on a Centricon filter (Amicon) with a 10-kDa cutoff to 20 mg of protein/ml. Preparation of [3H]TAG-loaded ER Enterocytes from your proximal half of male Sprague-Dawley rat small intestines were isolated and radiolabeled with [3H]oleate as explained (20). In brief, enterocytes were isolated from intestinal villi, collected, incubated with albumin-bound [3H]oleate for 30 min at purchase AZD2171 35 C, and washed with 2% BSA to remove the excess [3H]oleate. The labeled enterocytes were homogenized using a Parr bomb, and the ER was isolated using a sucrose step gradient, which was repeated to purify the ER (21). Isolation of a 75-kDa Protein Complex by Gel Filtration Chromatography 1 mg of cytosol was applied to a Sephacryl S-100 HR column (1.5 cm 45 cm) previously equilibrated with PBS (pH 7.2). The circulation rate was 0.5 ml/min, and the cytosol was eluted with PBS (pH 7.2) at 4 C. 1-ml fractions were collected. 3H disintegrations/min radioactivity was determined by liquid scintillation spectroscopy for each portion. For immunoblot, proteins in each portion were concentrated using a Millipore centrifugal Cspg2 filter unit (Millipore Corp., Billerica, MA) and suspended in Laemmli’s buffer. The presence of FABP1 in each portion was analyzed by immunoblot using anti-FABP1 antibodies.