Figure 4.

AP-1 and clathrin stability to Tris. (A) Duplicate binding assays using ARF·GTP (lanes 1–4), ARF1(Q71L)·GTP (lanes 5–8), or ARF1·GTPγS (lanes 9–12) to promote clathrin coat assembly were performed. One of the two membrane pellets was resuspended in 100 μl of the assay buffer, and the other was resuspended in 100 μl of 1 M Tris-HCl (pH 7.0). After incubation on ice for 10 min, the membranes were again collected, and the supernatants were individually aspirated and then precipitated with methanol/chloroform. The supernatant and pellet samples were resuspended in identical volumes of sample buffer and then analyzed by immunoblotting using the anti-clathrin heavy-chain (CHC) antibody and the anti-AP-1 μ1 subunit (μ1) antibody. AP and AS indicate the pellet and supernatant fractions, respectively, after reincubation with assay buffer; TP and TS denote the pellet and supernatant fractions, respectively, after reincubation with 1 M Tris-HCl (pH 7.0). (B) Quantitation of the μ1 subunit recovery in the pellet fraction after incubation in either acetate assay buffer (AP) or 1 M Tris-HCl, pH 7.0 (TP). The data are expressed as a ratio of the μ1 subunit immunoreactivity found in the Tris pellet compared with that found in the acetate pellet and are the mean ± SEM of four independent experiments. (C) Lanes 1–4 represent standard ARF1·GTP recruitment assays that were stripped with assay buffer or with 1 M Tris-HCl (pH 7.0), and lanes 5–8 represent assays treated similarly after recruitment with 50 μM AlCl₃ and 30 mM NaF also present. Bkg represents the background amount of AP-1 found in the pellet after incubation in the absence of nucleotide.