Journal: The Journal of reproduction and development

Article Title: Calyculin A-sensitive protein phosphatases are involved in maintenance of progressive movement in mouse spermatozoa in vitro by suppression of autophosphorylation of protein kinase A.

doi: 10.1262/jrd.20170

Figure Lengend Snippet: Fig. 1. Effects of incubation with protein phosphatase 1 (PP1)/protein phosphatase 2A (PP2A) inhibitor or cyclic adenosine 3’,5’- monophosphate (cAMP) analog on the protein phosphorylation state in mouse epididymal spermatozoa. Mouse epididymal spermatozoa were incubated with either a cell-permeable PP1/ PP2A inhibitor, calyculin A (CL-A, 25–125 nM), or a cAMP analog, Sp-5,6-DCI-cBiMPS (cBiMPS, 0.1 mM), at 37.5 C. In the left panels (each of the Western blots is representative of three replicates), aliquots of each sperm suspension (5.0 × 105 spermatozoa/lane) were recovered immediately before and after incubation for 90 or 180 min, used for SDS-PAGE and transblotting to the membranes and then treated with either the rabbit anti-phosphoserine (pS)/phosphothreonine (pT) PKA substrate polyclonal antibody (1:2,000, Anti-pS/pT PKA substrate) or mouse anti-phosphotyrosine (pY) monoclonal antibody (1:10,000, Anti-pY) and subsequently with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulins (1:2,000) or HRP-conjugated anti-mouse immunoglobulins (1:2,000). In the right panels (each photograph of indirect immunofluorescence is representative of three replicates), aliquots of each sperm suspension (5.0 × 105 spermatozoa/ preparation) were recovered immediately before and after incubation with either CL-A (125 nM) or cBiMPS (0.1 mM) for 90 or 180 min, smeared on glass slides, fixed with methanol and then treated with the anti-pS/pT PKA substrate antibody (1:50, Anti-pS/pT PKA substrate) and subsequently with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulins (1:25). The numbers on the photographs (0, 90 and 180) indicate the incubation time (min). In each set of photographs, the right photograph is a differential interference contrast image and the left photograph is an immunofluorescence image. The scale bar indicates 50 μm. Arrows indicate the principal pieces of sperm flagella.

Article Snippet: The primary antibodies were rabbit anti-phosphoserine (pS)/phosphothreonine (pT) PKA substrate polyclonal antibody [Cell Signaling Technology, Inc, Beverly, MA, USA, Cat. #9621, (this antibody recognizes amino acid sequences such as “arginine [R]-X-X-pS, R-X-X-pT and R-X-pS” and is a useful tool in identifying substrates of AGC family kinases, including PKA and protein kinase C; see the catalog of Cell Signaling Technology), 1:2,000], mouse anti-phosphotyrosine (pY) monoclonal antibody (Upstate Cell Solutions, Charlottesville, VA, USA, 4G10, Cat. #05-321, 1:10,000), rabbit anti-PKA catalytic subunit polyclonal antibody (Cell Signaling, Cat. #4782, 1:2,000), goat anti-PKA regulatory subunit II polyclonal antibody (Upstate, Cat. #06-411, 1:2,000), sheep anti-PP1γ2 polyclonal antibody (Exalpha Biologicals, MA, USA, Cat. #P130P, 1:2,000) and rabbit anti-phospho-PP1 (Thr-320) antibody (Cell Signaling, Cat. #2581, 1:2,000).

Techniques: Incubation, Phospho-proteomics, Western Blot, Suspension, SDS Page, Immunofluorescence

Journal: The Journal of reproduction and development

Article Title: Calyculin A-sensitive protein phosphatases are involved in maintenance of progressive movement in mouse spermatozoa in vitro by suppression of autophosphorylation of protein kinase A.

doi: 10.1262/jrd.20170

Figure Lengend Snippet: Fig. 3. Immunodetection of protein kinase A (PKA) and PP1γ2 in mouse epididymal spermatozoa. In each of the Western blots (representative of three replicates), aliquots of the sperm suspensions (5.0 × 105 spermatozoa/lane) were used for SDS-PAGE and transblotting to membranes and then treated with rabbit anti-PKA catalytic subunit polyclonal antibody (1:2,000, Anti-PKA catalytic subunit), goat anti-PKA regulatory subunit II polyclonal antibody (1:2,000, Anti-PKA regulatory subunit II) or sheep anti-PP1γ2 polyclonal antibody (1:2,000, Anti-PP1γ2) and subsequently with HRP-conjugated anti-rabbit immunoglobulins (1:2,000) or HRP-conjugated anti-goat immunoglobulins (1:2,000). In each set of photographs of indirect immunofluorescence (representative of three replicates), aliquots of each sperm suspension (5.0 × 105 spermatozoa/preparation) were smeared on glass slides, fixed with methanol and then treated with anti-PKA catalytic subunit antibody (1:50), anti-PKA regulatory subunit II antibody (1:50) or anti-PP1γ2 polyclonal antibody (1:50) and subsequently with FITC-conjugated anti-rabbit immunoglobulins (1:25) or FITC-conjugated anti-goat immunoglobulins (1:25). In each set of photographs of indirect immunofluorescence, the upper photograph is an immunofluorescence image and the lower photograph is a differential interference contrast image. The scale bar indicates 50 μm.

Article Snippet: The primary antibodies were rabbit anti-phosphoserine (pS)/phosphothreonine (pT) PKA substrate polyclonal antibody [Cell Signaling Technology, Inc, Beverly, MA, USA, Cat. #9621, (this antibody recognizes amino acid sequences such as “arginine [R]-X-X-pS, R-X-X-pT and R-X-pS” and is a useful tool in identifying substrates of AGC family kinases, including PKA and protein kinase C; see the catalog of Cell Signaling Technology), 1:2,000], mouse anti-phosphotyrosine (pY) monoclonal antibody (Upstate Cell Solutions, Charlottesville, VA, USA, 4G10, Cat. #05-321, 1:10,000), rabbit anti-PKA catalytic subunit polyclonal antibody (Cell Signaling, Cat. #4782, 1:2,000), goat anti-PKA regulatory subunit II polyclonal antibody (Upstate, Cat. #06-411, 1:2,000), sheep anti-PP1γ2 polyclonal antibody (Exalpha Biologicals, MA, USA, Cat. #P130P, 1:2,000) and rabbit anti-phospho-PP1 (Thr-320) antibody (Cell Signaling, Cat. #2581, 1:2,000).

Techniques: Immunodetection, Western Blot, SDS Page, Immunofluorescence, Suspension

Journal: The Journal of reproduction and development

Article Title: Calyculin A-sensitive protein phosphatases are involved in maintenance of progressive movement in mouse spermatozoa in vitro by suppression of autophosphorylation of protein kinase A.

doi: 10.1262/jrd.20170

Figure Lengend Snippet: Fig. 4. Co-immunoprecipitation of PP1γ2 with PKA in lysates from mouse epididymal spermatozoa. Mouse epididymal spermatozoa were treated with ice-cold lysis buffer. The resultant sperm lysates (sperm lysate: from 1.0 × 106 spermatozoa/lane) were used for immunoprecipitation using protein A-agarose beads with the anti-PKA regulatory subunit II polyclonal antibody (1:10, IP: anti-PKA RII) or without this antibody [IP: none (control)]. The precipitated fractions (precipitated fraction: from 2.0 × 106 spermatozoa/lane) were separated from the unprecipitated fractions (unprecipitated fraction: from 2.0 × 106 spermatozoa/lane) by centrifugation. The obtained samples were used for SDS-PAGE and Western blotting using anti-PP1γ2 polyclonal antibody (1:2,000, left panel; representative of three replicates) or anti-PKA regulatory subunit II polyclonal antibody (1:2,000, right panel; representative of three replicates).

Article Snippet: The primary antibodies were rabbit anti-phosphoserine (pS)/phosphothreonine (pT) PKA substrate polyclonal antibody [Cell Signaling Technology, Inc, Beverly, MA, USA, Cat. #9621, (this antibody recognizes amino acid sequences such as “arginine [R]-X-X-pS, R-X-X-pT and R-X-pS” and is a useful tool in identifying substrates of AGC family kinases, including PKA and protein kinase C; see the catalog of Cell Signaling Technology), 1:2,000], mouse anti-phosphotyrosine (pY) monoclonal antibody (Upstate Cell Solutions, Charlottesville, VA, USA, 4G10, Cat. #05-321, 1:10,000), rabbit anti-PKA catalytic subunit polyclonal antibody (Cell Signaling, Cat. #4782, 1:2,000), goat anti-PKA regulatory subunit II polyclonal antibody (Upstate, Cat. #06-411, 1:2,000), sheep anti-PP1γ2 polyclonal antibody (Exalpha Biologicals, MA, USA, Cat. #P130P, 1:2,000) and rabbit anti-phospho-PP1 (Thr-320) antibody (Cell Signaling, Cat. #2581, 1:2,000).

Techniques: Immunoprecipitation, Lysis, Control, Centrifugation, SDS Page, Western Blot

Journal: The Journal of reproduction and development

Article Title: Calyculin A-sensitive protein phosphatases are involved in maintenance of progressive movement in mouse spermatozoa in vitro by suppression of autophosphorylation of protein kinase A.

doi: 10.1262/jrd.20170

Figure Lengend Snippet: Fig. 5. Effects of incubation with PP1/PP2A inhibitor or cAMP analog on changes in the phosphorylation state of PP1 in mouse epididymal spermatozoa. Mouse epididymal spermatozoa were incubated with CL-A (125 nM) or cBiMPS (0.1 mM) at 37.5 C. Aliquots of each sperm suspension (5.0 × 105 spermatozoa/lane) were recovered immediately before and after incubation for 90 min, used for SDS-PAGE and transblotting to membranes and then treated with sheep anti-PP1γ2 polyclonal antibody (Anti- PP1γ2, 1:2,000) or rabbit anti-phospho-PP1 (Thr-320) polyclonal antibody [Anti-phospho-PP1 (Thr-320), 1:2,000] and subsequently with HRP-conjugated anti-goat immunoglobulins (1:2,000) or HRP-conjugated anti-rabbit immunoglobulins (1:2,000). Each blot is representative of three replicates.

Article Snippet: The primary antibodies were rabbit anti-phosphoserine (pS)/phosphothreonine (pT) PKA substrate polyclonal antibody [Cell Signaling Technology, Inc, Beverly, MA, USA, Cat. #9621, (this antibody recognizes amino acid sequences such as “arginine [R]-X-X-pS, R-X-X-pT and R-X-pS” and is a useful tool in identifying substrates of AGC family kinases, including PKA and protein kinase C; see the catalog of Cell Signaling Technology), 1:2,000], mouse anti-phosphotyrosine (pY) monoclonal antibody (Upstate Cell Solutions, Charlottesville, VA, USA, 4G10, Cat. #05-321, 1:10,000), rabbit anti-PKA catalytic subunit polyclonal antibody (Cell Signaling, Cat. #4782, 1:2,000), goat anti-PKA regulatory subunit II polyclonal antibody (Upstate, Cat. #06-411, 1:2,000), sheep anti-PP1γ2 polyclonal antibody (Exalpha Biologicals, MA, USA, Cat. #P130P, 1:2,000) and rabbit anti-phospho-PP1 (Thr-320) antibody (Cell Signaling, Cat. #2581, 1:2,000).

Techniques: Incubation, Phospho-proteomics, Suspension, SDS Page

Journal: The Journal of reproduction and development

Article Title: Calyculin A-sensitive protein phosphatases are involved in maintenance of progressive movement in mouse spermatozoa in vitro by suppression of autophosphorylation of protein kinase A.

doi: 10.1262/jrd.20170

Figure Lengend Snippet: Fig. 6. Effects of incubation with PP1/PP2A inhibitor or cAMP analog on the phosphorylation state at the Thr-197 of PKA catalytic subunit in mouse epididymal spermatozoa. Mouse epididymal spermatozoa were incubated with CL-A (125 nM) or cBiMPS (0.1 mM) at 37.5 C. Aliquots of each sperm suspension (5 × 105 spermatozoa/preparation) were recovered immediately before and after incubation for 90 min, smeared on glass slides, fixed with methanol and then treated with the rabbit anti-phospho- PKA (Thr-197) polyclonal antibody (1:50) and subsequently with FITC-conjugated anti-rabbit immunoglobulins (1:25). The numbers on the photographs (0 and 90) indicate the incubation time (min). In each set of photographs (representative of three replicates), the left photograph is an immunofluorescence image and the right photograph is a differential interference contrast image. The scale bar indicates 50 μm. Arrows indicate the principal pieces of sperm flagella.

Article Snippet: The primary antibodies were rabbit anti-phosphoserine (pS)/phosphothreonine (pT) PKA substrate polyclonal antibody [Cell Signaling Technology, Inc, Beverly, MA, USA, Cat. #9621, (this antibody recognizes amino acid sequences such as “arginine [R]-X-X-pS, R-X-X-pT and R-X-pS” and is a useful tool in identifying substrates of AGC family kinases, including PKA and protein kinase C; see the catalog of Cell Signaling Technology), 1:2,000], mouse anti-phosphotyrosine (pY) monoclonal antibody (Upstate Cell Solutions, Charlottesville, VA, USA, 4G10, Cat. #05-321, 1:10,000), rabbit anti-PKA catalytic subunit polyclonal antibody (Cell Signaling, Cat. #4782, 1:2,000), goat anti-PKA regulatory subunit II polyclonal antibody (Upstate, Cat. #06-411, 1:2,000), sheep anti-PP1γ2 polyclonal antibody (Exalpha Biologicals, MA, USA, Cat. #P130P, 1:2,000) and rabbit anti-phospho-PP1 (Thr-320) antibody (Cell Signaling, Cat. #2581, 1:2,000).

Techniques: Incubation, Phospho-proteomics, Suspension, Immunofluorescence