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. Author manuscript; available in PMC: 2014 Jan 24.
Published in final edited form as: Mol Cell. 2012 Nov 29;49(2):273–282. doi: 10.1016/j.molcel.2012.10.022

Figure 5. Functional Studies of PGAM5 in Cells.

Figure 5

(A) PGAM5(Δ24) binds XIAP and cIAP1 in vivo. On the left, HEK293 cells were cotransfected with Flag-cIAP1 and PGAM5(FL)-Myc or PGAM5(Δ24)-Myc. Total cell lysates were analyzed by immune-blotting with anti-Flag and anti-Myc antibodies (bottom two panels). Anti-Myc immune-precipitates (IP) were subjected to immunoblot analysis using anti-Myc and anti-Flag antibodies (top two panels). On the right, PGAM5(Δ24)-Myc was transiently expressed in HEK293 cells. 36 hours after transfection, cells were lysed and subjected for Immuno-precipitation with either anti-Myc antibody or an isotype control antibody (mouse IgG). Proteins in the whole cell lysate (bottom three panels) and proteins that bind to the antibody conjugated sepharose beads (top three panels) were assayed with immuno-blotting. *, indicates the position of the immunoglobulin heavy chain.

(B) XIAP and cIAP1 stimulate PGAM5(Δ24) ubiquitination in vivo. HeLa cells were transiently co-transfected with HA-Ubiquitin, PGAM5(Δ24)-Myc, with or without Flag-IAP construct. Ubiquitination of PGAM5(Δ24)-Myc were addressed by immuno-precipitating with Myc antibody then blotted with an HA antibody.

(C) Cytosolic PGAM5(Δ24) is increased in apoptotic cells. Jurkat and HeLa cells were treated with 1 μM STS for 4 hours. The cytosolic protein extracts were used for immuno-blotting analysis with anti-PGAM5 antibody.

(D) Time course of increasing PGAM5(Δ24) in cytosol. Jurkat cells were treated with 1 μM STS and harvested at different time points to extract cytosolic proteins for immuno-blotting analysis with anti-PGAM5 antibody.

(E) High PGAM5 expression induces apoptosis. Jurkat cells were co-transfected with indicated PGAM5 constructs and GFP at a 5:1 molar ratio. 24 hours after transfection, cell viability was measured by Annexin V staining. Different expression levels of GFP were gated to indicate the expression of PGAM5. Error bars represent SEM calculated from triplicate experiments. Representative anti-Myc immuno-blotting from one of the three experiments shows the relative expression levels of PGAM5(FL) and PGAM5(Δ24).

(F) PGAM5 sensitizes cells to the apoptosis inducer STS. HeLa cells are transiently transfected with indicated PGAM5 constructs in 96 well format. 24 hours after transfection, cells are treated for 2 hours with STS at different concentrations. Cell viability is then immediately measured by CellTiter-Glo luminescent assay. Percentages of apoptotic cells are normalized with DMSO treated samples. PGAM5 levels are analyzed with anti-Myc immune-blotting before STS treatment. Error bars represent SEM calculated from triplicate samples.

See Figure S4.