Methods for yeast two-hybrid screening of a human testis cDNA library using human PPP1CC have been previously described [15, 48, 49]. DNA sequence analysis was performed using an ABI PRISM 310 Genetic Analyser (Portugal Applied Biosystems, Porto, Portugal). The DNA sequences obtained were compared to the NCBI database, using the BLAST algorithm (http://BLAST.ncbi.nlm.nih.gov/). The multiple sequence alignments were performed using the ClustalW program from Ensembl.
PPP1R2 and PPP1R2P3 cloning, expression and purification
The cDNA of PPP1R2P3 was ligated into the pET28c (Novagen, Madison, Wisconsin, USA) expression vector using EcoRI and XhoI restriction sites, adding a histidine tag (His-tag) to the N-terminus of the protein. The pET-PPP1R2P3 sequence was verified and the plasmid transformed into E. coli strain Rosetta (DE3) (Novagen, Madison, Wisconsin, USA). The expression of His-tag PPP1R2P3 was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hrs at 37ºC and the protein purified using a Ni-NTA resin (QIAGEN, Dusseldorf, Germany) according to the supplier’s instructions. Briefly, the cells were lysed in 10 mM imidazole, sodium phosphate buffer, pH 8.0, centrifuged at 15000 g for 30 min at 4ºC, and the supernatant was applied to the resin. The resin was washed with 20 mM imidazole and the His-tag PPP1R2P3 was eluted with 500 mM imidazole. The protein was further purified by 12% SDS-PAGE. A portion of the lane containing PPP1R2P3 was stained with Coomassie blue and the region containing the remaining protein was excised, washed three times with water, cut into smaller pieces and 1 ml of 100 mM Tris-HCl, pH 8.5, 0.1% SDS was added before freezing at -20ºC overnight. The slurry was frozen and thawed three times and then passed through a 0.22 μm filter membrane. The gel-free filtrate was then dialyzed against 4 × 500 ml of 10 mM Tris-HCl, pH 7.5 buffer for 24 hrs at 4ºC. The protein concentration of recombinant His-PPP1R2P3 was determined by BCA® assay (Fisher Scientific, Loures, Portugal). PPP1R2P3 cDNA was also ligated into the pTACTAC expression vector  in the NdeI and XbaI restriction sites, the sequence verified and then transformed into Rosetta strain. The expression of PPP1R2P3 was induced with 0.4 mM IPTG for 3 hrs at 37ºC. The protein was partially purified by boiling the bacterial extract (here on referred to as recombinant PPP1R2P3) as previously described for PPP1R2 . PPP1R2 cDNA was likewise ligated in pTACTAC expression vector  and recombinant protein purified in the same way as for PPP1R2P3 (here on referred to as recombinant PPP1R2). PPP1R2P3 cDNA was excised using EcoRI and XhoI from pET-PPP1R2P3 and subcloned in pACT-2 vector (Clontech, Saint Germain-en-Laye, France) for co-transformation.
Yeast co-transformation with plasmid DNA
Yeast competent AH109 cells were co-transformed with pACT-PPP1R2P3 and pAS2-PPP1CA, pAS2-PPP1CC1, pAS2-PPP1CC2 or pAS2-PPP1CC2end, by the lithium acetate method . pAS2-PPP1CC constructs used were previously described . For negative and positive controls pAS2-1/pACT-2 and pVA3-1/pTD1-1 vectors were used, respectively. Afterwards, the transformation mixture was plated on selective media containing X-α-Gal and incubated at 30ºC to check for MEL1 expression as indicated by the appearance of a blue color (Clontech, Saint Germain-en-Laye, France).
Blot overlay analysis
For blot overlay analysis, 0.3 μg of commercial PPP1R2 (NEB, New England Biolabs, Herts, UK) and recombinant His-PPP1R2P3 were resolved by SDS-PAGE and then transferred to a nitrocellulose membrane. Blots were overlaid with purified PPP1CC1 or PPP1CC2 (25 pmol/mL) diluted in Tris buffered saline with Tween-20/BSA [48, 51] and detected with the antibodies CBC3C (against the C-terminal of PPP1CC, which detects both isoforms ) or CBC502 (specific for the C-terminal of PPP1CC2), both raised in rabbit. Immunoreactive bands were revealed by incubating with horseradish peroxidase conjugated anti-rabbit secondary antibody and developed by enhanced chemiluminescence (ECL, GE Healthcare, Amersham Biosciences Europe GmbH, Freiburg, Germany).
Phosphatase activity assays
The IC50 values of PPP1R2 and PPP1R2P3 for purified PPP1CC1 and PPP1CC2 isoforms were determined using [32P]phosphorylase a as substrate. The substrate was prepared from phosphorylase b (Sigma-Aldrich Química, S.A., Sintra, Portugal) using [γ-32P]ATP (3000 Ci/mmol, GE Healthcare, Amersham Biosciences Europe GmbH, Freiburg, Germany) and phosphorylase kinase (Sigma-Aldrich Química, S.A., Sintra, Portugal) as previously described . An appropriate range of concentrations of commercial PPP1R2 and His-PPP1R2P3 were incubated with the purified PPP1C isoforms and the phosphatase activity determined. The IC50 was calculated using the BioDataFit 1.02 software (Chang Bioscience, Castro Valley, California, USA).
Ejaculated sperm were collected from healthy donors by masturbation into an appropriate sterile container. Spermograms were performed by experienced technicians and only samples with normal parameters were used . For all the methods, sperm was washed three times in 1× PBS. For immunoprecipitation, sperm was lysed in 1× RIPA buffer (radioimmunoprecipitation buffer, Millipore Iberica S.A.U., Madrid, Spain) supplemented with protease (10 mM benzamidine, 1.5 μM aprotinin, 5 μM pepstatin A, 2 μM leupeptin, 1 mM PMSF) and phosphatase (1 mM sodium fluoride, 2.5 mM sodium pyrophosphate, 50 mM beta-glycerophosphate, 1 mM sodium orthovanadate) inhibitors, sonicated 3× for 10 sec and centrifuged at 16000 g for 20 min, at 4ºC. The supernatant was collected and heat-stable extracts were prepared by immersing the sample in a boiling water bath for 30 min, chilled on ice for 2 min and centrifuged at 16000 g for 20 min, at 4ºC. The final supernatant was used in the subsequent steps. For Western blot, both the supernatant (soluble fraction) and the pellet (insoluble fraction) were resuspended in 1% SDS .
For the preparation of heads and tails, washed sperm were briefly sonicated and the detachment checked by phase contrast microscopy (PH) using an Olympus IX81 epifluorescence microscope, equipped with appropriate software (Olympus Portugal - Opto-Digital Tecnologias, S.A., Lisboa, Portugal). Sperm were directly applied onto a sucrose step gradient (1.8 M, 2.02 M and 2.2 M) to separate heads from tails, by centrifuging at 5000 g for 1 hr and fractions corresponding to tails and heads collected. Subsequently fractions were centrifuged at 16000 g, 10 min, resulting in a pellet free of sucrose and proteins were dissolved in 1% SDS. For immunocytochemistry, washed sperm was used directly on the coverslips. Human testicular biopsy was also prepared using 1% SDS and the same protocol as for sperm samples, but previously homogenized using a tissue homogenizer. Testicular biopsy was collected in Centro Hospitalar de Coimbra, Portugal during a procedure to collect organs for transplantation from a brain death 35-years-old adult man and the biopsy was diagnosed as “normal spermatogenesis” based on histopathological analysis.
Written informed consent, for the use of sperm samples, was obtained from the donors for publication and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. The study was conducted in accordance with the guidelines of the “Helsinki Declaration”.
Testicular biopsies for research purposes are covered by the legislation of the Portuguese Constitution (decreto-lei nº274/99 of July 22, 1999: “It is permitted the dissection of corpses, or parts of them, of national citizens, stateless persons and foreign residents in Portugal, as well as extraction of parts, tissues or organs when the deceased has explicitly declared in life's will that his body can be used for purposes of teaching and scientific research.”).
Phosphorylation of PPP1R2 and PPP1R2P3
Recombinant PPP1R2, PPP1R2P3 or human sperm heat extract of native PPP1R2/PPP1R2P3 (about 200 ηg of inhibitor protein in 50 mM Tris-HCl pH 7.5, 0.1 mM EGTA and 0.03% Brij-35) were phosphorylated by GSK3β or CK2 (Calbiochem, MERCK, Darmstadt, Germany) or both kinases as previously described . The phosphorylation reaction was run at 30ºC for 90 min and then terminated with the addition of 4× SDS loading buffer. Two separate 12% gels were run, one with 1/10 of the reaction volume and the other with the remaining 9/10 of the reaction volume. The 1/10 reaction gel was transferred and analyzed by Western blot while the 9/10 reaction gel was dried and autoradiographed.
RIPA supernatant sperm extracts were pre-cleared using dynabeads protein G (Life Technologies S.A., Madrid, Spain). A direct immunoprecipitation approach was performed with 1 μg of sheep anti-PPP1R2 or rabbit anti-PPP1R2 pre-incubated with Dynabeads® Protein G during 1 hr at 4ºC with rotation. After incubation, pre-cleared sperm extracts were applied to the antibody-dynabeads complex and incubated overnight with rotation at 4ºC. After washing three times with 1× PBS in 3% BSA for 10 min with rotation at 4ºC, beads were resuspended in loading buffer and boiled.
For mass spectrometry analysis, immunoprecipitates were resolved by 10% SDS-PAGE along with purified positive controls. Gels were stained with Coomassie blue colloidal (Sigma-Aldrich Química, S.A., Sintra, Portugal). In brief, gels were fixed with a fixation solution (40% methanol and 10% acetic acid) during 1 hr, washed with distilled water and then transferred to the Coomassie blue colloidal staining solution for 1 hr. After staining, the gels were washed with distilled water and afterwards destained with 25% methanol until bands were visualized.
Bands were excised directly from the gel using a spatula and completely destained. In-gel digestion was performed overnight at 37°C with trypsin (Promega, Madison, Wisconsin, USA) in 10 mM HCl and 50 mM ammonium hydrogen carbonate (NH4HCO3) at pH 7.8. Resulting peptides were extracted once with 100 μl of 1% formic acid (FA), and twice with 100 μl of 5% FA, 50% acetonitrile (ACN). Extracts were combined and ACN was removed in vacuo. For LC-MS analysis, a final volume of 40 μl was prepared by addition of 1% FA. Electrospray tandem mass spectrometry (ESI-MS/MS) was performed on an Orbitrap Velos instrument (Thermo Scientific, Bremen, Germany). Fragment ions were generated by low-energy collision-induced dissociation (CID) on isolated ions with a fragmentation amplitude of 0.5 V. MS spectra were summed from four individual scans ranging from m/z 300–1500 with a scanning speed of 8.100 (m/z)/s. MS/MS spectra were a sum of two scans ranging from m/z 100–2800 at a scan rate of 26.000 (m/z)/s. Generated data were imported to ProteinScape™, a proteomics data platform (Bruker Daltonik GmbH, Bremen, Germany) and analyzed using MASCOT (version 2.2.0, Matrix Science, London, UK) search algorithm with search parameters as follows: precursor ion tolerance of 1.2 and 0.3 Da for MS/MS spectra. Proteins were considered to be identified if the Mascot score (ProteinScape™) was higher than 65.
Extracts were mass normalized using BCA® assay (Fisher Scientific, Loures, Portugal). Immunoprecipitates and extracts were resolved by 10% SDS-PAGE. Proteins were subsequently electrotransferred onto nitrocellulose membranes and immunodetected with the appropriate antibodies, using ECL detection (GE Healthcare Spain, Madrid, Spain). The primary antibodies used in this study included sheep polyclonal anti-PPP1R2 (1:100), the rabbit CBC502 (1:2000, against the C-terminal of PPP1CC2), the rabbit CBC3C (1:1000, against the C-terminal and detects both PPP1CC isoforms) and the loading controls, mouse monoclonal anti-β-tubulin (1:500, Life Technologies S.A., Madrid, Spain) and mouse monoclonal acetylated-α-tubulin (1:2000, Life Technologies S.A., Madrid, Spain). The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit (1:5000), anti-sheep (1:1000) and anti-mouse (1:5000) IgGs for ECL detection (GE Healthcare, Amersham Biosciences Europe GmbH, Freiburg, Germany).
An aliquot of washed diluted sperm (25 μl) was placed onto a glass coverslip pre-coated with 100 μg/ml poly-L-ornithine, and dried at room temperature, in a six-well plate containing one coverslip per well. To each well 1 ml of 4% paraformaldehyde in 1× PBS was gently added and left to stand for 10 min. Subsequently, sperm sample was washed twice with 1 ml 1× PBS for 10 min. For permeabilization, 1 ml of 1:1 methanol/acetone solution was added for 2 min and then the samples washed twice with 1 ml 1× PBS for 10 min and blocked for 1 hr with 3% BSA in 1× PBS, before incubation with primary antibodies (rabbit CBC502, 1:250 and sheep anti-PPP1R2, 1:100) for 2 hrs at room temperature. After three washes with 1× PBS, the fluorescently labeled secondary antibodies anti-rabbit Texas-Red, 1:300 (MolecularProbes, Eugene, USA) and anti-sheep FITC, 1:50 (DAKO, Glostrup, Denmark) were added and the coverslips incubated for 2 hrs. Finally, three washes with 1× PBS were performed and coverslips were mounted on microscope glass slides with one drop of anti-fading reagent containing DAPI for nucleic acid staining (Vectashield, Vector Laboratories Burlingame, California, USA). Images were acquired using an Olympus IX81 epifluorescence microscope and digital camera, equipped with the appropriate software (Olympus Portugal-Opto-Digital Tecnologias, S.A., Lisboa, Portugal).
The human sperm heat-stable extracts (hsPPP1R2) or hsPPP1R2 plus 10 ηg of recombinant PPP1R2 were acetone precipitated and the pellets were resuspended in 250 μl of 2D rehydration solution (8 M Urea/ 2 M Thiourea/ 2% CHAPS/ 0.002% of bromophenol blue) and supplemented with 2.5 μl of IPG buffer (in the 4–7 pH range) and 14 mg of DTT. The samples were pipetted into a strip holder and the electrophoresis was started (1 hr at 30 V, 2 hrs at 150 V, 1 hr at 500 V, 1 hr at 1000 V and 2 hrs at 8000 V). After the second dimension on 12% gels, samples were analyzed by Western blot.
Dephosphorylation of human sperm PPP1R2/PPP1R2P3
The hsPPP1R2 extracts were incubated overnight with either protein tyrosine phosphatase 1B (Upstate, Millipore Iberica S.A.U., Madrid, Spain) at 37ºC, calf intestinal phosphatase (NEB, New England Biolabs (UK), Herts, UK) at 37ºC or PPP1CC1, at 30ºC, with the respective assay buffers. The reactions were stopped with the addition of 4× SDS loading buffer and analyzed by Western blot.