Fig. 2. Cellular distribution of PIX and PAK. (A) Distribution of PIX and PAK by cell fractionation. COS-7 cells transfected with pXJ-Flag-ß1PIX and pXJ-Flag-ß2PIX were fractionated, analysed by SDS-PAGE and probed with anti-Flag antibodies. Untransfected COS-7 cell fractions were also analysed by SDS-PAGE and probed with anti-PIX-SH3 or anti-/ß-PAK (PAK1 and Pak3) antibodies. The fractions collected are: T, total protein; S, water-soluble proteins (S100 fraction); Tx, Triton-X-100-soluble proteins (P100); P, SDS-soluble proteins of the pellet from Triton-X-100 extraction, which contain cytoskeletal proteins. (B) Cellular localization of PIX isoforms and mutants. Various PIX constructs were transiently transfected into HeLa cells. The DNA constructs used were pXJ-Flag-PIX, pXJ-Flag-ß1PIX, pXJ-Flag-ß2PIX, pXJ-Flag-ß1PIX^1-555, pXJ-Flag-N80ß1PIX and pXJ-GST-ß1PIX-C-ter. The proteins expressed were immunostained using anti-Flag or anti-GST antibodies. Constructs that contained the coiled-coil C terminus were localized at the cell periphery, whereas ß2PIX and the C-terminally truncated mutant ß1PIX^1-555 showed cytoplasmic and nuclear staining.