Figure 1.
SHP protein is localized on the mitochondrial outer membrane.
(A) Western blots to detect Flag-mSHP, VADC, COXIV, HSP60, and Cyto C proteins using specific antibodies for each protein, with the exception of SHP, which was detected with an anti-Flag antibody. Huh7 cells were transfected with Flag-mSHP (20 µg, 15 cm plate), and five plates were used to harvest protein. Differential centrifugation was used to isolate the mitochondria-enriched precipitate fraction (P1), microsome-enriched precipitate fraction (P2), and cytosolic supernatant fraction (S); the whole cell lysate (W) was used for comparison. (B) Alkaline digestion of mitochondrial membrane fraction (Pm) and soluble fraction (Sm) and Western blots to determine Flag-mSHP, VADC, COXIV, HSP60, and Cyto C proteins. (C) Trypsin protection assays and Western blots to determine Flag-mSHP, VADC, Bcl2, and Cyto C proteins in mitochondria. Abbreviations: mito, mitochondria; mem, membrane; solu, soluble; peri, peripheral; pt, protein.
Figure 2.
Deletion of the N-terminal fragment increases SHP protein levels in mitochondria.
(A) Diagram showing the deletion mutation constructs of Flag-hSHP. FL, full length; ND, N-terminal deletion. (B) Left, Western blots to determine SHP protein expression in different cellular fractions in Huh7 cells. Right, band intensities were measured by densitometry, and the intensities relative to that of the whole cell lysate (set as 1) were plotted. Data are represented as mean ± SEM (*, P<0.01). Abbreviations: whole, whole cell lysate protein; nu, nuclear protein; mito, mitochondria protein. PARP is a marker for nuclear proteins; VDAC is expressed in mitochondria.
Figure 3.
SHP interacts with Bcl2 primarily through its interaction domain in vitro.
(A) Diagram showing GST-mSHP deletion constructs. (B) Left, GST pull-down assay to determine the in vitro interaction of SHP with Bcl2. GST or GST-mSHP full-length or deletion mutants used in the reactions were indicated by asterisks. The interaction of Bcl2 with GST-mSHP proteins was detected by Western blots using a Bcl2 antibody. Right, band intensities were measured by densitometry and normalized to each corresponding GST-mSHP fusion protein. Data are represented as mean ± SEM (#, P<0.01). Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.
Figure 4.
The transmembrane domain of Bcl2 is critical for its interaction with SHP.
(A) Diagram showing the domain structure of Bcl2 protein. (B) Top, GST pull-down assay to determine the interaction domain of Bcl2 with SHP and the effect of AHPN on the in vitro interaction of Bcl2 with SHP. Bcl2wt, ΔTM, ΔBH1, ΔBH2, and ΔBH3 proteins were detected using an antibody against the N-terminus of Bcl2. ΔBH4 and Δloop plasmids contain a myc epitope, and these proteins were detected using an anti-Myc antibody. Bottom, Bcl2 band intensities were measured by densitometry, and the values were normalized by dividing by the GST-SHP Int intensities. The values for intensities relative to Bcl2 wt (set as 1) were plotted. Data are represented as mean ± SEM (*, P<0.01). Abbreviations: BH, BH domain; TM, transmembrane domain.
Figure 5.
Ectopic expression of HNF4α induces an exclusive SHP nuclear translocation.
(A) The subcellular localization of GFP-hSHP in 293T cells was observed under a fluorescence microscope, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 100 µm, 50 µm (insets). (B) The N-terminal domain of SHP is primarily responsible for the interaction with HNF4α protein. Left, Diagram showing the deletion mutation constructs of GFP-mShp. Right, 293T cells were transfected with GFP-mSHP deletion constructs (4 µg, 6 cm plate) along with HA-HNF4α (4 µg, 6 cm plate) expression vector. Anti-HA agarose was used to immuneprecipitate HNF4α, and the protein levels of various GFP-mSHP truncation mutants and HNF4α were detected by Western blots using anti-GFP or anti-HA antibodies, respectively. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.
Figure 6.
SHP nuclear translocation depends on its N-terminal domain.
The subcellular localization of GFP-mSHP full length and deletion mutants in 293T cells was observed by fluorescence microscopy, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 50 µm. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.