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Figure 8


Fig. 8. ORP3 is phosphorylated depending on the cell-adhesion status. (A) HEK293 cells were cultured in normal medium for 20 hours. Confluent adherent (AD) cells were lyzed directly on the plate or trypsinized briefly and cells were allowed to adhere and spread on the fibronectin-coated plate in normal medium for 1 hour (replated cells, RP). Adherent and replated cells were treated with 100 nM phorbol myristate acetate (PMA) or 10 µM all-trans retinoic acid (ATRA) for 60 minutes before lysis. (B) A time course in which trypsinized cells were replated and allowed to adhere and spread for 20-120 minutes (indicated at the top). (C) Cells treated as specified above were lyzed and treated without and with {lambda}-phosphatase (indicated at the bottom). (D) Confluent adherent cells treated with 50 nM okadaic acid for the indicated times. PMA-treated adherent cell sample was used as a positive control for the band shift to the upper position. Quantified intensities of the two ORP3 bands (arrows) are indicated at the bottom. (E) Trypsinized cells (Trypsin) washed and lyzed directly in SDS-PAGE loading buffer or (F) incubated for 1 hour on a dish treated with denatured BSA to inhibit adhesion (DenBSA). Adherent and replated cells are shown for a comparison. (G) Comparison of adherent and replated cells with those at 40-60% confluency (AD Subconfluent). (H) Adherent cells were treated with 5 mM EGTA (AD EGTA) or E-cadherin-blocking antibodies (AD DECMA-1) for 30 minutes.





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