Figure 5. A ternary protein complex... | Rockefeller University Press

$Figure 5. A ternary protein complex comprised of Gimap5, HSC70, and Mcl-1 or Bcl-xL. (A) Cell lysates from parental 70Z/3 cells (parental) or 70Z/3 cells that stably express Flag-tagged Gimap5 (Gimap5nFL) were incubated with Sepharose beads conjugated with anti-Flag. The immune complex was subjected to Western blot analysis with the indicated antibodies. (B) Cell lysates from parental or Gimap5nFL 70Z/3 cells were incubated with Sepharose beads conjugated with anti-Flag. The immune complex was subjected to SDS-PAGE and Coomassie blue staining. Proteins specifically immunoprecipitated with anti-Flag in cell lysates derived from Gimap5nFL 70Z/3 cells, but not parental 70Z/3 cells, were subjected to an in-gel tryptic digestion. The peptides derived from the proteins were subjected to MALDI-TOF mass spectrometry analysis. (C) Cell lysates from parental or Gimap5nFL 70Z/3 cells were immunoprecipitated with anti–Mcl-1 or anti–Bcl-xL or control IgG. The immune complex was subjected to Western blot analysis with the indicated antibodies. (D) Cell lysates from parental or Gimap5nFL 70Z/3 cells were immunoprecipitated with anti-HSC70. The immune complex was subjected to Western blot analysis with the indicated antibodies. (E) The cytosol and mitochondria fractions were extracted from parental or Gimap5nFL 70Z/3 cells and subjected to Western blot analysis with the indicated antibodies. Tom20 was used as a marker of mitochondria fraction. (F) Gimap5nFL 70Z/3 cells were applied to glass slides and subjected to immunofluoresence analyses with anti-Flag (green) with a combination of anti–Mcl-1 or anti-HSC70 (red). Data shown are representative of three (A and E) or two (C, D, and F) independent experiments.$

Figure 5.

A ternary protein complex comprised of Gimap5, HSC70, and Mcl-1 or Bcl-xL. (A) Cell lysates from parental 70Z/3 cells (parental) or 70Z/3 cells that stably express Flag-tagged Gimap5 (Gimap5nFL) were incubated with Sepharose beads conjugated with anti-Flag. The immune complex was subjected to Western blot analysis with the indicated antibodies. (B) Cell lysates from parental or Gimap5nFL 70Z/3 cells were incubated with Sepharose beads conjugated with anti-Flag. The immune complex was subjected to SDS-PAGE and Coomassie blue staining. Proteins specifically immunoprecipitated with anti-Flag in cell lysates derived from Gimap5nFL 70Z/3 cells, but not parental 70Z/3 cells, were subjected to an in-gel tryptic digestion. The peptides derived from the proteins were subjected to MALDI-TOF mass spectrometry analysis. (C) Cell lysates from parental or Gimap5nFL 70Z/3 cells were immunoprecipitated with anti–Mcl-1 or anti–Bcl-xL or control IgG. The immune complex was subjected to Western blot analysis with the indicated antibodies. (D) Cell lysates from parental or Gimap5nFL 70Z/3 cells were immunoprecipitated with anti-HSC70. The immune complex was subjected to Western blot analysis with the indicated antibodies. (E) The cytosol and mitochondria fractions were extracted from parental or Gimap5nFL 70Z/3 cells and subjected to Western blot analysis with the indicated antibodies. Tom20 was used as a marker of mitochondria fraction. (F) Gimap5nFL 70Z/3 cells were applied to glass slides and subjected to immunofluoresence analyses with anti-Flag (green) with a combination of anti–Mcl-1 or anti-HSC70 (red). Data shown are representative of three (A and E) or two (C, D, and F) independent experiments.

This Feature Is Available To Subscribers Only