Arf6 copurifies with the peroxisomal membrane fractions. (A) Rat liver homogenates were fractionated into a postnuclear (E), a nuclear (N), a heavy mitochondrial (M), a light mitochondrial (L), a microsomal (P), and a cytosolic (S) fraction. A peroxisome-enriched fraction (PO) was obtained by centrifugation of the L-fraction on a Nycodenz step gradient (see Methods). Equal amounts of protein were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies raised against the nuclear pore complex protein p62 (Nup62), mitochondrial glutamate dehydrogenase (GDH), lysosomal-associated membrane protein 2 (LAMP2), the ER-resident chaperone immunoglobulin binding protein (BiP/GRP78), the microtubule-binding peripheral Golgi membrane 58 kDa protein (Golgi 58 K), the plasma membrane protein pan-cadherin (Pan-cadherin), the peroxisomal membrane protein Pex13p (Pex13p), peroxisomal biogenesis factor 19 (Pex19p), or a linear epitope found in Arf proteins (1D9). Note that the majority of Golgi 58 K is soluble after fractionation, and that Pex19p is a predominantly cytosolic, partly peroxisomal protein. (B) Six milligrams of total protein from the peroxisomal fraction was processed for floatation centrifugation in an alkaline sucrose gradient (see Methods). The fractions were collected from the bottom, processed for SDS-PAGE, transferred to a nitrocellulose membrane, and stained for total protein with Ponceau S (upper panel). A select set of fractions was immunoblotted with anti-Arf 1D9 or antibodies against Arf6 or peroxisomal matrix (catalase), core (urate oxidase), or membrane proteins (Pex3p, PMP70) (lower panels). The density (in g/ml) of the gradient fractions and the migration of the molecular mass markers (their masses expressed in kDa) are indicated.