Fig. 4. Immunoprecipitation assays. (A) CHO_Vß₃ cells and (B) osteoclast cultures were treated as described in Fig. 2, immunoprecipitated with the indicated antibodies and western blotted for PKC (diluted 1:800), FAK (diluted 1:1000), PYK2 (diluted 1:500), ERK-2 and ß₃ detection (left and middle panels). Total cell lysates were also subjected to SDS-PAGE and western blotted for total FAK and PYK2 detection in CHO_Vß₃ cells (A, right panels) and osteoclasts (B, right panels). Similar results were obtained in three independent preparations. IP, immunoprecipitation; PI, preimmune serum; TCL, total cell lysate; WB, western blot analysis; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. (C) An osteoclast plated in an LM609-coated well was immunostained with anti-PKC (diluted 1:10) and _V (diluted 1:10) antibodies detected by conventional immunofluorescence using TRITC- (diluted 1:200) and FITC- (diluted 1:160) conjugated secondary antibody, respectively. Note that the osteoclast was allowed to adhere for 30 minutes, a time insufficient to permit actin ring formation. Scale bars: 20 µm (upper panels); 5 µm (lower panels). Brackets in the upper panels delineate the area showed at higher magnification in the lower panels. Arrows indicate areas of overlapping red (PKC) and green (_V) fluorescent labels.