Fig. 3. Localization of NG2 to cellular retraction fibers. Parental B28 cells (g,h) and B28 cells expressing wild-type NG2 (clone 6, a,b), NG2/S1342A (clone 42, c,d), and NG2/S999A (clone 51, e,f) were grown overnight on poly L-lysine-coated plates, fixed with 2% paraformaldehyde, and then double-labeled with monoclonal anti-NG2 antibody (b,d,f,,h) and rabbit antibody against whole B28 cells (a,c,e,g). The paraformaldehyde fixation is important because antibody incubations and repeated washing of living cells tend to induce cell ‘rounding’, accompanied by retraction fiber formation. Thus, the fixed cells give a more accurate picture of cell morphology under actual culture conditions. In the case of cells expressing wild-type NG2 and NG2/S1342A, abundant retraction fibers visible by staining with the poly-specific B28 antiserum are also heavily labeled by the anti-NG2 antibody. B28 cells and transfected cells expressing NG2/S999A have many fewer retraction fibers. In the NG2/S999A transfectants these fibers are not well stained by the anti-NG2 antibody, as seen previously in Fig. 2. B28 cells are negative for NG2. Bar, 10 µm (h).
