Expression of ILP-2. (A) Northern blot analysis of human multiple-tissue blots and cell lines (Clontech) was performed using high-stringency conditions and a ³²P-labeled cDNA fragment of full-length ILP-2. The ∼8.5-kb band present in all lanes corresponds to ILP-1, which cross-reacts with the ILP-2 probe. As a loading control, the blot was probed for β-actin (below). Sizes (in kilobase) are indicated. (B) RT-PCR of total RNA from human tissues of heart, testis, and lung using ILP-2-specific oligonucleotide primers and reverse transcriptase (top). As a negative control, PCRs were performed without reverse transcriptase (middle). A control PCR using GAPDH-specific primers and reverse transcriptase amplified equal amounts of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) PCR fragment of the RNA of each tissue (bottom).

ILP-2 inhibits caspase activation in a cell-free system. (A, B) Cytosolic extracts (150 μg) from 293 cells were preincubated with GST–ILP-1, GST–ILP-2, or GST control proteins (100 ng each) as indicated for 20 min, at which time ATP (1 mM final concentration) was added and the reaction mixture was incubated for a further 30 min. (C, D) Cytosolic extracts were incubated with ATP for 30 min, after which time the recombinant GST–ILP-1, GST–ILP-2, or GST control proteins were added and the reaction mixture was incubated for a further 20 min. Caspase activity was measured by cleavage of the caspase 3-specific fluorogenic substrate DEVD-AFC (A, C) or the caspase 9-specific substrate LEHD-AFC (B, D), as described in Materials and Methods.

ILP-1 and ILP-2 inhibit processing of caspases 9 and 3 in a cell-free system. 293 cell extracts (1.5 mg) were incubated with recombinant, purified GST–ILP-1, GST–ILP-2 or control GST proteins (1 μg) for 30 min, activated with ATP for 30 min, and resolved by gel filtration chromatography on a Superose 6 column (Amersham Pharmacia) using an ÄKTA FPLC system (Amersham Pharmacia). Aliquots of the fractions eluted from the column were separated by SDS-PAGE, immunoblotted onto nitrocellulose, and probed with the indicated antibodies as follows. (A) Apaf-1 (Y. Lazebnik, Cold Spring Harbor Laboratories, N.Y.). The calculated molecular mass of Apaf-1 was consistent with the expected molecular mass of 130 kDa and is indicated (p130). (B) Caspase 9 (Upstate Biotechnology). The full-length (Pro) and processed 35- and 37-kDa species (p35/37) are indi- cated. (C) Caspase 3 (Pharmingen). The inactive precursor (Pro) and the 19- and 17-kDa processed, active forms (p19/17) are indicated. (D) Aliquots of the fractions were also evaluated for DEVDase activity, using DEVD-AFC (20 μM) as substrate as described in Materials and Methods. Molecular masses (in kilodaltons) are shown.

ILP-2 is conserved during the evolution of great apes. (A) Schematic representation of ILP-1 and ILP-2. (B) Sequence comparison of the ILP-1 and ILP-2 proteins. The predicted amino acid sequences of the corresponding region of human ILP-1 (residues 262 to 497) are shown aligned with the full coding sequence of human ILP-2, chimpanzee ILP-2, and gorilla ILP-2. The GenBank accession number of human ILP-2 is AF164682, and the accession numbers of chimpanzee and gorilla ILP-2 are AY030052 and AY030053, respectively. (C) Southern blot analysis of EcoRI-digested genomic DNA from humans, great apes (chimpanzee and gorilla), Old World monkeys (baboon, cynomolgus monkey, and rhesus monkey), and mouse (C57BL6) was obtained by using a probe which detects both ILP-1 and ILP-2. Included in the experiment were the human genomic clones of ILP-1 (PAC–ILP-1) and ILP-2 (BAC–ILP-2). The gorilla ILP-2 gene does not contain an EcoRI site in its coding sequence, accounting for a profile that differs slightly from those of the human and chimpanzee genes.

ILP-2 inhibits apoptosis induced by Bax and caspase 9 plus Apaf-1 but not by Fas. Human embryonic kidney 293 cells were transfected with a GFP plasmid (0.5 μg/well) in combination with either a Bax expression plasmid (0.25 μg/well) (A), Fas expression plasmid (2 μg/well) (B), or Apaf-1 (2 μg/well) and caspase 9 (0.5 μg/well) plasmids, as indicated (C). Cells were cotransfected with a plasmid encoding GFP as a marker of transfection, together with a control plasmid or plasmids expressing ILP-1, ILP-2, or dominant-negative caspase 9 (C-9 DN) (2 μg/well) as indicated. Cells were fixed and stained with DAPI, and transfected wells were scored for viability based on nuclear morphology as described in Materials and Methods. Data shown are representative of at least three independent experiments.

ILP-1 (Δ2BIR) and ILP-2 interact with processed caspase 9 in cells. 293 cells were transfected with expression vectors encoding GST, GST–ILP-1, GST–ILP-2, and/or caspase 9 as indicated (2 μg/well) (A), GST–ILP-2, caspase 9, and/or caspase 9 mutants as indi- cated (B), and GST–ILP-1, GST–ILP-1 (Δ2BIR), GST–ILP-2, and/or caspase 9 as indicated (C). Cell lysates were coprecipitated with glutathione-Sepharose beads, washed, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with an anti-caspase 9 antibody (Pharmingen). The full-length (Pro) and processed (p35/37) forms of caspase 9 are indicated. Cell lysates were also examined by immunoblot analysis to confirm transfection efficiency and loading (input). The bands marked with asterisks are found only when the GST–ILP-1 vector is coexpressed with caspase 9 and presumably represent the caspase 9-induced cleavage products which have been reported previously (9, 23). Molecular masses (in kilodaltons) are shown.