Fig. 10. The 9-kDa protein is an ER resident protein and its phosphorylation is reduced by PKC removal or inhibition. (A) Lane 1: PCTV-budding reaction including 20 µCi [³²P-P] ATP (see Fig. 1A) was performed using native cytosol. Lane 2: a similar PCTV-budding reaction was performed using PKC-immunodepleted cytosol and ER washed with 2 M urea. Lane 3: PCTV-budding assay was performed similar to that shown in lane 1, except that 4 µg of calphostin C were pre-incubated with the cytosol. Lane 4: PCTV-budding assay was performed as that shown in lane1 using cytosol treated with 50 µM pseudosubstrate. The reaction was stopped by placing the incubation tubes on ice and ading cold HEPES buffer (10 mM, pH 7.2). Of the reaction 30 µl were solubilized in Laemmli buffer and the proteins separated on an 8%-16% gradient SDS-PAGE. Autoradiography was performed on the separated proteins (10 days at –70°C). Only the band at 9 kDa is shown. (B) A PCTV-budding reaction supplemented with 50 µCi [–³²P]ATP was performed and the reaction components were separated on a continuous sucrose gradient. PCTV were collected from the top of the gradient, ER from the bottom and the remaining fractions were taken as cytosol. 30 µg protein from each fraction (cytosolic proteins were solubilized from a TCA precipitate) were separated by 8%-16% gradient SDS-PAGE and autoradiography was performed on the separated proteins (30 hours at –70°C). Lane 1, ER; lane 2, cytosol; lane 3, PCTV. Only the band at 9 kDa is shown. (C) Lane 1: result from intestinal ER (500 µg) and cytosol (1 mg) incubated with [-³²P]ATP and 2 mM NaF as in Fig. 9A and an autoradiogram was performed after 40 µg of the proteins had been separated by 8%-16% SDS-PAGE. Lane 2: ER proteins (2 mg) were incubated with rPKC (5 µg) and [-³²P]ATP for 30 minutes at 37°C in the presence of 2 mM NaF. The ER was isolated, washed and 40 µg of the proteins were separated by 8%-16% SDS-PAGE. The gel was dried and the phosphorylation of ER proteins determined by autoradiography.