Methyl-b-cyclodextrin, filipin, cytochalasin-D, dynasore monohydrate, and 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) were from Sigma. Ammonium chloride and concanamycin-A were from Fluka. Latrunculin-A, and Alexa-Fluor labelled human transferrin, dextran and phalloidin were from Invitrogen. Stock solutions of Methyl-b-cyclodextrin, transferrin and dextran were made in GMEM, and ammonium chloride in sterile water. Stock solutions of phalloidin were made in methanol. Stock solutions of other inhibitors were made in dimethyl sulfoxide (DMSO). Where appropriate, an equivalent dilution of DMSO (or methanol) was included in the mock treatment. Cells were seeded on glass coverslips (BDH) in antibiotic-free cell-culture medium and transfected when ,60% confluent. Cells were transfected in Optimem (Invitrogen) using a ratio of 1 mg plasmid DNA to 1 ml Lipofectamine 2000 (Invitrogen) according to the manufacturers guidelines. Cells were incubated at 37uC, 5% CO 2 for 4 h when the transfection medium was replaced with antibiotic-free cell-culture medium. Cells were used for experiments at 12 h post-transfection. Cells expressing a transgene were identified by confocal microscopy. Transfection efficiencies (determined by GFP or c-myc expression) ranged from 30-43%.