Bacterial Expression and Cell Culture Protocol

All bacteria were cultured in Luria–Bertani medium with appropriate antibiotics at 37°C. Plasmids were maintained in E. coli XL-1 Blue (Stratagene, San Diego, CA) and purified using standard miniprep kits (Machery-Nagel, Bethlehem, PA) before transfection. During bacterial expression, GST-fusion proteins were produced in E. coli Rosetta(DE3)pLysS (EMD Millipore, Billerica, MA). Sf9 insect cells were grown in ESF921 medium (Expression Systems, Davis, CA) at 28°C and infected with baculoviruses encoding MBP- or GST-fusion proteins at a multiplicity of infection of ∼1. Cos7, HeLa, and HFF cells were cultured in DMEM plus 10% fetal bovine serum and antibiotic-antimycotic (Invitrogen) at 37°C in 5% CO₂. Transfections with DNA or RNA used Lipofectamine-LTX or RNAiMAX (Invitrogen), respectively (Campellone et al., 2008 (link)). Control siRNAs, siRNAs to glyceraldehyde-3-phosphate dehydrogenase, and siRNA pair #3 to Rab1a+b were from Ambion, and siRNA pairs #1 and 2 to Rab1a+b and siRNAs #1 and 2 to RhoD were from Sigma-Aldrich (St. Louis, MO). For selection of HFF clones stably transfected with LAP or LAP-WHAMM, media were supplemented with 750 μg/ml G418. To induce LAP expression, 7.6 mM sodium butyrate was added for 12–18 h.