Fig 1.
Immunoreactivity of mouse ADAM2 antibodies.
Lysates from testicular cells and sperm from wild-type and Adam2-/- mice were boiled in 3% SDS and 5% β-mercaptoethanol and subjected to Western blot analyses. A. Western blotting with the anti-mADAM2-D antibody. This antibody was raised against the ADAM2 disintegrin domain, and recognized the precursor (100-kDa) and processed (45-kDa) forms of ADAM2 in mouse testis and sperm, respectively. B and C. Blots performed using two different antibodies, which were raised against the cytoplasmic tail domain and designated B. anti-mADAM2-CyT-1 and C. anti-mADAM2-CyT-2. D. An antibody against a-tubulin (TUBA) was included as a control. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviations: T, testicular cells; S, sperm; WT, wild-type; A2-/-, Adam2-/-; NS, non-specific. Molecular masses are presented on the left.
Fig 2.
Coimmunoprecipitation of ADAM2 in mouse testes.
A and B. Testes from wild-type and Adam2-/- mice were lysed with 1% NP-40 buffer, the tissue lysates were immunoprecipitated with A. anti-mADAM2-CyT-1 and B. anti-mADAM2-CyT-2, and the resolved immunoprecipitates were immunoblotted with anti-mADAM2-D. Normal rabbit serum was used as a control. C and D. Lysates were precipitated with C. anti-mADAM2-Cyt-1 and D. anti-mADAM2-CyT-2 and analyzed by immunoblotting with anti-mADAM2-D, anti-mADAM1B, and anti-mADAM3. Experiments were repeated five times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gels. Abbreviations: WT, wild-type; A2-/-, Adam2-/; TL, tissue lysate (100 μg); IP, immunoprecipitated protein (1 mg); NRS, normal rabbit serum; and IB, immunoblotting.
Fig 3.
Amino acid sequence comparison of the mouse, monkey and human ADAM2 proteins.
A. amino acid sequence identities are given as human versus mouse and monkey. B. Comparison of amino acid sequences of mammalian ADAM2. The consensus residues are shaded. Abbreviations: Pro, prodomain; Metallo, metalloprotease domain; Dis, disintegrin domain; Cys, cysteine-rich domain; EGF, epidermal growth factor (EFG)-like domain; T, transmembrane domain; Cy, cytoplasmic tail domain; Ag-1, antigen region of the anti-hADAM2-Cys antibody; and Ag-II, antigen region of the anti-hADAM2-CyT antibody.
Fig 4.
Specificity of the generated antibodies against human ADAM2.
A and C. Testicular germ cells were isolated from monkey testis, boiled in 3% SDS and 5% β-mercaptoethanol, subjected to SDS-PAGE, and blotted with A. anti-hADAM2-Cys and C. anti-hADAM2-CyT antibodies. B and D. Human testis was lysed with 0.1% NP-40 buffer, and Western blot analysis was performed using B. anti-hADAM2-Cys and D. anti-hADAM2-CyT. Glutathione S-transferase or GST recombinant ADAM2 (GST-hADAM2-Cys and GST-hADAM2-CyT) proteins were added to the buffer containing the primary antibodies during Western blot analysis. E and G. The supernatants from the monkey testis lysates were boiled in 3% SDS with (lane 1) or without (lanes 2 and 3) 5% β-mercaptoethanol, and blotted with E. anti-hADAM2-Cys and G. anti-hADAM2-CyT. F and H Human testis supernatants were boiled in 3% SDS and with (lane 1) or without (lanes 2 and 3) 5% β-mercaptoethanol, and immunoblotted with F. anti-hADAM2-Cys and H. anti-hADAM2-CyT. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gels. Abbreviation: GST, glutathione S-transferase; β-ME, β-mercaptoethanol. Molecular masses are presented on the left.
Fig 5.
Expression of ADAM2 in monkey and human germ cells.
A and B. Extracts of testicular cells and sperm from A. monkeys and B. humans were subjected to SDS-PAGE, and blotted with anti-hADAM2-Cys. C and D. Lysates from C monkey and D human germ cells were immunoblotted with anti-hADAM2-CyT. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviation: T, testicular cells; S, sperm; and β-ME, β-mercaptoethanol. Molecular masses are presented on the left.
Fig 6.
Biochemical characteristics of ADAM2 in monkey and human sperm.
A and C. Monkey sperm were boiled in 3% SDS with (lane 1) or without (lanes 2 and 3) 5% β-mercaptoethanol, resolved by SDS-PAGE and blotted with A. anti-hADAM2-Cys and C. anti-hADAM2-CyT. The 47-kDa band in the β-mercaptoethanol-free sample was exposed to mercaptoethanol diffusing from the adjacent lane (left). Thus, it is mostly reduced on the left side of the lane and mostly non-reduced on the right side of the lane. B and D. Human sperm were boiled in 3% SDS with (lane 1) or without (lanes 2 and 3) 5% β-mercaptoethanol, resolved by SDS-PAGE and blotted with B. anti-hADAM2-Cys and D. anti-hADAM2-CyT. Experiments were repeated twice. Reduced and non-reduced samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviation: β-ME, β-mercaptoethanol. Molecular masses are presented on the left.
Fig 7.
Integrity of human sperm proteins.
Western blot analysis was performed using antibodies against ADAM2, PRKACA, PRKARIα, HSPA5, and ADAM7. The TUBA and GAPDH antibodies were used as controls. Experiments were repeated twice. Reduced protein samples were subjected to SDS-PAGE using 10% resolving gels. Abbreviation: NS, non-specific band. Molecular masses are presented on the left.
Table 1.
Previous proteomic analyses.
Fig 8.
Immunoprecipitation with anti-hADAM2-CyT.
Immunoprecipitation of ADAM2 was carried out using monkey testis lysates. Immunoprecipitation using normal rabbit serum was performed as a negative control. Immunoprecipitated lysates were immunoblotted with anti-hADAM2-CyT. Experiments were repeated three times. Reduced protein samples were subjected to SDS-PAGE using 8% resolving gel. Abbreviations: TL, tissue lysate (100 μg); S, supernatant; and IP, immunoprecipitated protein (1 mg); and NRS, normal rabbit serum.
Table 2.
Monkey ADAM2-coimmunoprecipitated proteins identified by LC-MS/MS analysis.
Fig 9.
Possible ADAM2 complexes in monkey and human testicular cells.
A and C. The non-reduced supernatants from the lysates of monkey testis were either boiled in 3% SDS (lane 2) or incubated at room temperature in 0.3% SDS (lane 1), and then immunoblotted with A. anti-hADAM2-Cys and C. anti-hADAM2-CyT. B and D. A similar experiment was performed using extracts from human testis immunoblotted with B. anti-hADAM2-Cys and D. anti-hADAM2-CyT. Asterisks and arrowheads indicate the ADAM2 complexes and monomers, respectively. Experiments were repeated three times. Reduced and non-reduced potein samples were subjected to SDS-PAGE using 8% resolving gels. Abbreviation: β-ME, β-mercaptoethanol. Molecular masses are presented on the left.