Mouse anti myc

HEK293 cells were cultured at 37°C in DMEM (Hyclone) supplemented with 10% FBS (Gibco) and 100 U/ml of penicillin G and streptomycin (Gibco). For transient transfection, cells were transfected with indicated plasmids using polyethylenimine (Sigma-Aldrich). In brief, cells were cultured in 6-well plates to 50% confluence, and then incubated with precipitates formed by 3 μg of plasmid DNA and 1 μl of polyethylenimine (5 mg/mL). After 8~12 h of incubation with PEI/DNA polyplex, media was replaced with the fresh serum-containing medium. At 48 h after initial transfection, cells were fixed with 4% PFA (paraformaldehyde) with 4% sucrose at room temperature for 15 min, permeabilized with 0.3% TritonTX-100, and blocked with 10% goat serum and 0.1% saponin. Primary antibodies used were rabbit anti-Flag (Sigma, 1:1,000), and mouse anti-myc (Developmental Studies Hybridoma Bank (DSHB), 1:1000). The colocalization was quantified using ImageJ software.

COS7 fibroblasts were maintained in DMEM supplemented with 10% FBS, pen/strep, L-glutamine at 37°C and 5% CO₂. Cells were transiently transfected with 1–2 µg of plasmid using Lipofectamine 2000 according to manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). For immunohistochemistry, cells were plated on Fibronectin-coated cover slips at a density of 4×10⁵ cells and allowed to grow for 18–24 hours. Cells were washed with PBS, fixed with either −20°C MeOH or 4% PFA, and stained as previously described (Hildebrand, 2005 (link)). The following antibodies were used: rabbit anti-Shroom3 (1:200, UPT132, (Hildebrand, 2005 (link)), mouse anti-myc (1:200, 9E10, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), mouse anti-Flag (1:200, Sigma, St Louis, MO, USA), and Alexa 488 or 568-conjugated secondary antibodies (1:400, Invitrogen). Cells were imaged using an Olympus Fluoview 1000 confocal microscope equipped with a 40× oil immersion objective (Olympus America, Center Valley, PA, USA). Two-dimensional projections were generated from z-series (0.5 µm steps) and images were processed using ImageJ.