Journal: Cell Adhesion & Migration

Article Title: Src-inducible association of CrkL with procaspase-8 promotes cell migration

doi: 10.4161/cam.25284

Figure Lengend Snippet: Figure 1. Identification of an interaction between Caspase-8 and Crk proteins. (A) Serum starved NB7 (Casp8−/−) and NB7C8 (Casp8 reconstituted) cells were trypsinized and held in suspension (Susp) or allowed to attach to fibronectin for 10, 30, or 60 min and lysates probed with indicated antibodies. (B) NB7C8 cells were subjected to immunoprecipitation with antisera to caspase-8 after spreading on a fibronectin substrate for 30 min (FN), or after being held in suspension (Susp) and probed for indicated proteins. (C) Similarly treated NB7C8 cells were subjected to Crk protein-immunoprecipitation and immunoblotting for the proteins indicated. NB7 cells with genetic deletion of caspase-8 were used as controls to clearly differentiate the procaspase-8 band from precipitating Ig. (D) Immobilized recombinant SH2 domains of Crk-I/II, CrkL or Syk were probed with recombinant caspase-8 catalytic domain treated with recombinant Src kinase (phosphocasp-8) or not (casp-8) (mean ± SE, *P < 0.01). (E) Recombinant SH2 domains of Crk-II and CrkL were used in pulldown assays, and the precipitants resolved and probed for indicated proteins. (F) Lysates prepared as in (A) expressing control shRNA or shRNA against CrkL. (G) NB7 or NB7C8 cells expressing CrkL shRNA or control shRNA were analyzed for their ability to migrate in a wound assay for four hours (n = 40/group, mean ± SE, *P < 0.01).

Article Snippet: Cells were stained with polyclonal antibody to procaspase-8 (BD Biosciences) (1:100) or mouse anti-CrkL antibody (BD Biosciences) (1:100) for 2 h. After multiple PBS/BSA washes, cells were exposed to respective Alexa Fluor conjugated secondary antibodies (1:250).

Techniques: Immunoprecipitation, Western Blot, Recombinant, Expressing, shRNA

Journal: Cell Adhesion & Migration

Article Title: Src-inducible association of CrkL with procaspase-8 promotes cell migration

doi: 10.4161/cam.25284

Figure Lengend Snippet: Figure 2. CrkL promotes recruitment of caspase-8 to the periphery. (A) NB7C8 cells expressing CrkL shRNA or control shRNA were allowed to spread on fibronectin coated coverslips, then fixed cells were stained for procaspase-8 and with phalloidin to identify actin-rich adhesion ruffles, then imaged by confocal microscopy. Representative images are shown. Scale bars indicate 10 μm. (B) Recruitment of caspase-8 to adhesion ruffles was evaluated (n = 20/group, mean ± SE, *P < 0.01). (C) NB7C8 cells were allowed to attach to fibronectin-coated plates for 30 min before sequential isolation of cytosolic or focal adhesion complex containing fractions and immunoblot analysis. Bars indicate ratio of caspase-8 to FAK (C8:FAK) as normalized to “control” shRNA, as determined by densitometry via ImageJ software from NIH (n = 2/group, mean ± SE, *P < 0.05).

Article Snippet: Cells were stained with polyclonal antibody to procaspase-8 (BD Biosciences) (1:100) or mouse anti-CrkL antibody (BD Biosciences) (1:100) for 2 h. After multiple PBS/BSA washes, cells were exposed to respective Alexa Fluor conjugated secondary antibodies (1:250).

Techniques: Expressing, shRNA, Staining, Confocal Microscopy, Isolation, Western Blot, Software