Figure 2.

Analysis of blebbishields for GSK3-β, p70S6K, 4EBP1, stem-cell transcription factors, and apoptotic signatures. (a) Effects of combined GSK-3β and p70S6K inhibition and DNA-damage (p70S6K cleavage) were studied by western blotting. The blebbishield and blebbishield-depleted fractions were isolated as described in Materials and methods. Lanes 3 and 4 are fractions of lane 2. Note the cleavages of 4EBP1 and p70S6K and phosphorylation of GSK-3β in blebbishields. (b) Characterization of blebbishields for the expression of core stem-cell transcription factors by western blotting. Note that N-myc and c-myc are downregulated in blebbishields. (c) Evaluation of caspase activation in blebbishields by western blotting. Caspase-8 Asp-391/372 was detected by an antibody specific to cleavage site, and the constitutively activated p18 fragment was detected by anti-p18 antibody (not specific to cleavage site). Note the inhibition of caspase-9 processing and induction of caspase-3 p17 in blebbishields. (d) Examining the role of caspases and granzyme-B in blebbishields. Note that the proportion of non-glycosylated granzyme-B versus glycosylated granzyme-B differed between blebbishields and blebbishield-depleted fraction (top panel). Fas but not FasL was expressed in blebbishields (Note that FasL was expressed in blebbishield-depleted fraction). PARP cleavage fragments in blebbishields indicating the involvement of both caspases and granzyme-B in blebbishield generation. MEM, minimal essential medium; NEAA, non-essential amino acid (except ℒ-glutamine); Vit., vitamins; Cisp, cisplatin