Radioactive Labeling and Immunoprecipitation of Proteins

Radioactive labeling of cells was performed on subconfluent cultured cells grown on 90-mm plates. For the ³³P-labeling, A431 cells were first incubated for 1 h at 37°C in serum-free and phosphate-free medium (Sigma Chemical Co.) before adding 0.5 mCi of ³³P-orthophosphate (Amersham, UK) to each 90-mm plate. After further incubation for 1 h at 37°C, cells were stimulated with 200 ng/ml EGF for different times up to 15 min. Cells were then quickly washed with TBS and scraped into 1 ml of extraction buffer containing phosphatase and protease inhibitors: 25 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 1 mM PMSF, a protease inhibitor cocktail (complete™, Boehringer), 1 mM Na₃VO₄, 30 mM NaF, and 10 mM Na₂P₂O₇. Similarly for ³⁵S-labeling, A431 and NRK cells grown on 90-mm plates were preincubated for 1 h at 37°C in methionine/cysteine-free MEM containing 5% dialysed FCS. After a brief wash with PBS, 0.2 mCi of [³⁵S]methionine/cysteine (Pro-mix; Amersham, UK) in 4 ml of methionine/cysteine free MEM supplemented with 20 mM Hepes, pH 7.4, was added to the cells and incubation continued for 1 h at 37°C. The cells were washed three times with PBS and scraped into 1 ml of extraction buffer without the phosphatase inhibitors. The cell extracts after ³⁵S- or ³³P-labeling were first sonicated on ice to reduce viscosity and then cleared by centrifugation at 50,000 g for 30 min. 200 μl of supernatant was mixed first with 20 μl of nonimmune rabbit serum and 20 μl of protein A–Sepharose beads (50 mg/ml; Sigma Chemical Co.) and incubated at 4°C for 30 min. The nonimmune complex was sedimented by centrifugation and the resultant supernatant incubated with 20 μl of the specific antiserum. After 1 h this mixture was centrifuged in a microfuge (13,000 g, 15 min) and the resultant supernatant mixed with 50 μl of protein A–Sepharose beads (50 mg/ml). After 1 h the immunoprecipitates were washed eight times with extraction buffer by briefly sedimenting in the microfuge, aspirating the supernatant and then resuspending the Sepharose beads again in 0.5 ml of extraction buffer. Finally the Sepharose beads was washed twice in 50 mM Tris-HCl, pH 7.4, before processing for SDS-PAGE.
For limited proteolytic cleavage of myosin VI, A431 cells were labeled as described above with ³³P. They were then extracted with a buffer containing 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, protease and phosphatase inhibitors (see above), and 1% IGEPAL CA-630 (Sigma Chemical Co.) but no denaturing detergent, because digestion with chymotrypsin in the presence of SDS leads to complete cleavage to small fragments. After immunoprecipitation and washing, pellets were finally washed and resuspended in 25 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1 mM CaCl₂, and incubated with Chymotrypsin (Sigma Chemical Co.) at 1:200 (wt/wt) for 30 min at 25°C. The digests were then processed for SDS-PAGE.