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Fig. 4 | Molecular Cancer

Fig. 4

From: The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1

Fig. 4

PS is essential for DLC1 inhibition of proliferation and migration. A Pull-down (PD) using control (ctrl) or PS-coated beads in 293 T overexpressing GFP DLC1 START 848–1091 and GFP or the C2 domain of Lactadherin (GFP-LACT-C2), followed by Western blot (WB) using an antibody that recognizes the C-terminal domain of DLC1 (DLC1 C-term). Input (I) represents a fraction of the protein lysates before pull-down. The correct expression of all plasmids in total lysates was checked by GFP Western blot (bottom panel). B Caveolin-1 or PLCD1 Immunoprecipitation (IP) in same cell lysates from A, followed by Western blot (WB) with the indicated antibodies. C Stable H358 control or DLC1-overexpressing cells were transfected with GFP or the C2 domain of Lactadherin (LACT-C2). 24 h after transfection, cells were plated at the same density and allow to proliferate for the indicated times (left panel) and counted, or to migrate in transwells overnight (middle panel). Migrated cells were fixed and photographed, and the number of migrated cells was quantified using Image J, from a total of four images taken from each experiment duplicate. The correct expression of all plasmids was checked by Western blot (WB) (right panel). D Parental CHO or PS-deficient mutant cell lines (PSA3 and PSB2) were transfected with GFP control or GFP-DLC1. 24 h after transfection, all cells were plated at the same density and allow to proliferate for 30 h (left panel) and counted, or to migrate in transwells overnight (middle panel). Migrated cells were fixed and photographed, and the number of migrated cells was quantified using Image J, from a total of four images taken from each experiment duplicate. Bars represent mean +/− SD. The percentage (%) of inhibition was calculated for DLC1 transfected cells compared to GFP control transfected cells, for each cell line. The correct expression of all plasmids was checked by GFP Western blot (WB) (right panel)

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