Mouse monoclonalanti eea1

We used a clone of PC12 cells, PC12 Nsc-1 (Cellomics Inc., Maryland, USA) cells, due
to their increased growth rate and decreased cell clumping, which facilitate imaging
experiments (Hahn et al., 2009 (link)). Cells were
grown following the manufacturer's instructions. PC12 cells were starved for
36 hr before stimulation either with 100 ng/ml EGF (Invitrogen) or 50 ng/ml NGF
(R&D Systems) for 30 min. E14.5 Dlk1+ hepatoblasts were starved for 24
hr before stimulation with either 10 ng/ml EGF or HGF (R&D systems). Then,
cells were fixed with 3% para-formaldehyde and stained with a mouse monoclonal
anti-EEA1 (BD Biosciences Pharmingen). A fluorescently conjugated goat
anti-mouse-AlexaFluor 555 secondary antibody (Molecular Probes, Invitrogen) revealed
the antigen signal. Image acquisition and image analysis were performed as described
above.

Immunostaining was performed as previously described9 (link). Cells were cultured on collagen-coated cover slips in a 12-well plate, fixed with 3.7% paraformaldehyde in PBS for 20 min, permealized with 0.2% (w/v) Triton X-100 in PBS for 10 min, and blocked with 1% (w/v) BSA/1% (w/v) gelatin in PBS. Primary antibodies used in cell staining were mouse monoclonal anti-FLAG M2 clone (Sigma, F1804), rabbit polyclonal anti-ATP7A (Hycult Biotech, HP8040), mouse monoclonal anti-ATP7A (Santa Cruz, 376467), mouse monoclonal anti-EEA1 (BD Biosciences, 610456), mouse monoclonal anti-Rab11 (BD Biosciences, 610656), mouse monoclonal anti-Lamp1 (developmental studies hybridoma bank, H4A3-s), and sheep monoclonal anti-TGN46 (Gene Tex, GTX74290). Secondary antibodies used for cell staining were: goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001), goat polyclonal anti-rabbit IgG Alexa555-conjugate (Molecular Probes, A-21428). Cells were imaged with a Zeiss LSM 710. Images were processed with ZEN and Image J. Anti-EEA1, anti-Rab11, and anti-Lamp1 antibodies were kindly provided by Dr Rajini Rao (Johns Hopkins University). All antibodies were used at a dilution of 1:200.