Sds22, PPP1R11, and PP1γ2 are bound to each other in crude testis protein extracts.

<p><i>A</i>. The recombinant proteins GST-PPP1R11 and control GST were incubated with testis cell lysates in the presence of Glutathione-Sepharose beads. The eluted proteins and testis extracts alone were resolved by SDS-PAGE and subjected to western blot analysis with anti-Sds22 and anti-PP1γ2 antibodies. <i>B</i>. Testis protein extracts and buffer controls were incubated with anti-PP1γ2, anti-PPP1R11, or preimmune serum immobilized on Protein G-Sepharose-4 beads, as indicated at the top of the figure. The immunoprecipitates were separated by SDS-PAGE and immunoblotted for the proteins indicated at the bottom of the figure. (ND: not done).</p

PP1γ2 is inactive in chromatographically-purified fractions containing PP1γ2, Sds22, PPP1R11, and actin.

<p><i>A</i>. Protein phosphatase activity in 5-µl aliquots of DEAE-, MonoQ-, and Superose 6-purified fractions from cauda sperm protein extracts. <i>B</i>. SDS-PAGE/immunoblot analysis of the corresponding fractions probed with anti-Sds22, anti-PP1γ2, anti-PPP1R11, anti-PP2A, and anti-actin antibodies.</p

PPP1R11 is present in various tissues including testis.

<p><i>A</i>. Validation of PPP1R11 antibody was performed as follows: recombinant PPP1R11 and testis protein extracts were separated by SDS-PAGE followed by western blot analysis with affinity purified rabbit polyclonal anti-PPP1R11. Size markers (left) were derived from β-Galactosidase (116-kDa), Phosphorylase b (97.4-kDa), Albumin (66-kDa), Glutamic dehydrogenase (55-kDa), Ovalbumin (45-kDa), Glyceraldehyde-3-phosphate dehydrogenase (36-kDa), Carbonic anhydrase (29-kDa), and Soybean trypsin inhibitor (20-kDa). <i>B</i>. Soluble protein extracts from testis and somatic tissues were separated by SDS-PAGE followed by western blot analysis with the validated affinity purified PPP1R11 antibody, and the same blot was stripped and reprobed with anti-actin used as a loading control.</p

Increasing steady state levels of PPP1R11, PP1γ2 and Sds22 in testis parallel the temporal progression of spermiogenesis.

<p>In each experiment, postnatal testis levels of PP1γ2, Sds22, and PPP1R11 were monitored by assessing equal concentrations of protein (25 µg) from extracts prepared from each age group. Proteins were separated by SDS-PAGE, and gels were assayed by western blotting with antibodies against PP1γ2, Sds22, and PPP1R11. Results indicating that postnatal expression of PP1γ2, Sds22, and PPP1R11 increased over the age range in which the initial round of spermiogenesis takes place were visualized by chemiluminescence. A duplicate blot was probed with an anti-actin antibody to demonstrate equal protein loading.</p

In PP1γ-null testis, PPP1R11 is ubiquitinated or associated with an ubiquitinated protein whose apparent size is equivalent to that of PPP1R11.

<p><i>A</i>. Western blot analysis of wild-type and PP1γ-null testis protein extracts probed with either anti-mono- and poly-ubiquitinylated conjugates or anti-PPP1R11 antibodies, as indicated below the blots. <i>B</i>. Western blot of anti-PPP1R11 or preimmune serum immunoprecipitates from testis extracts probed with a monoclonal antibody that recognizes mono- and poly-ubiquitinylated conjugates.</p

PPP1R11, Sds22 and PP1γ2 co-elute during chromatographic purification of testis extracts.

<p>Western blot analysis of purified column fractions shows a consistent co-elution pattern of testicular PP1γ2, Sds22, and PPP1R11 through a series of chromatographic media. <i>A</i>. DEAE chromatography showing PP1γ2, Sds22, and PPP1R11 co-eluting primarily in fractions A3 and A4. <i>B</i>. A3 and A4 fractions from the DEAE column were pooled and purified by MonoQ column fractionation. PP1γ2, Sds22, and PPP1R11 co-eluted in fractions B8–11. <i>C</i>. B8–B11 fractions from the MonoQ column were pooled and purified by Superose 6 column chromatography, from which PP1γ2, Sds22, and PPP1R11 co-eluted from 14.05–15.05 ml. In each instance 0.5 ml of co-eluting fractions were collected and concentrated, then assayed by SDS-PAGE/western blot analysis.</p

Immunolocalization of the Na⁺,K⁺-ATPase and arrestin in the presence of PP2A in COS cells.

<p>COS cells were transfected with H85N plus Na⁺,K⁺-ATPase β-subunit (A), with H85N, Na⁺,K⁺-ATPase β-subunit and flag tagged arrestin (B-D), with H85N, Na⁺,K⁺-ATPase β-subunit and HA tagged PP2A C-subunit (E-G), or with H85N plus Na⁺,K⁺-ATPase β-subunit and flag tagged arrestin plus HA tagged PP2A C-subunit (H-M). Cells were stained with HK9 (A, B, E, H and K), anti-flag for arrestin (C and I), and anti-HA for PP2A C-subunit (F and L) antibodies. Overlay patterns are shown in D, G, J and M. (×200 magnification) A large fraction of the H85N was found in intracellular compartments when cells expressed arrestin in the absence of PP2A C-subunit. This effect was not observed when arrestin was expressed together with the PP2A C-subunit. Typical results from one of three experiments are shown.</p

Testicular phenotypes related to the expression of PP1γ2.

<p><i>A</i>. Immunofluorescence of PP1γ-null (KO), wild-type (WT), and PP1γ2-rescue testis sections probed with anti-PPP1R11 antibody. Negative (−) control = no primary antibody. <i>B</i>. Histological analysis of hematoxalin-stained testis sections from PP1γ-null (Null) and PP1γ2-rescue (Rescue) mice demonstrating an antiapoptotic effect of PP1γ2. <i>C</i>. Western blot of testis protein extracts from PP1γ-null (left lane), PP1γ2-rescue (center lane), and wild-type (right lane) animals probed with anti-Sds22, anti-PP1γ2, anti-PPP1R11, and anti-actin antibodies. <i>D</i>. Upper panel: PPP1R11 level in PP1γ-null spleen is not significantly diminished compared to its level in wild-type spleen, but is significantly diminished in tissues normally expressing PP1γ2 (testis and brain); Lower panel: histogram of immunosignal ratios of PPP1R11 in PP1γ-null vs wild-type tissues. <i>E</i>. Northern blot analysis showing that <i>Ppp1r11</i> mRNA levels are comparable in PP1γ-null and wild-type brain and testis, respectively. Actin mRNA levels are used as gel loading control. <i>F</i>. Left panel: PP1γ isoform levels are increased and PPP1R11 levels rise dramatically in PP1α-null vs wild-type testis and brain, but PP1γ1 and PPP1R11 levels in PP1α-null spleen are unchanged; Right panel: histogram of immunosignal ratios of PPP1R11 in PP1α-null vs wild-type tissues.</p

Actin co-precipitates with PP1γ2, Sds22, and PPP1R11 in testis.

<p>Fractions from mouse testis protein extracts first purified by DEAE-, MonoQ-, and Superdex 200-column chromatography were immunoprecipitated with anti-actin, anti-PPP1R11, anti-Sds22 and anti-PP1γ2 antibodies. Following SDS-PAGE of the immunoprecipitates, the gels were analyzed by western blotting with antibodies against the proteins indicated to the right of the blots.</p

PPP1R11, Sds22, and PP1γ2 from co-eluting fractions are reciprocally co-immunoprecipitated, and co-migrate by native PAGE.

<p><i>A</i>. Superose 6 fractions (C4 and C5 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004861#pone-0004861-g003" target="_blank">Figure 3C</a>) of testis extracts containing co-eluted PPP1R11, PP1γ2, and Sds22 were incubated with anti-PPP1R11, anti-PP1γ2, anti-Sds22, or preimmune serum immobilized on Protein G-Sepharose 4 beads, as indicated at the top of the figure. The immunoprecipitates were separated by SDS-PAGE and immunoblotted for co-precipitating proteins as indicated below each blot strip (The reason why Sds22 migrates as a doublet is not known). <i>B</i>. C4 and C5 fractions of testis proteins purified by Superose 6 column chromatography were also separated by native PAGE followed by western blot analysis. Triplicated blot strips probed with either anti-PPP1R11, anti-PP1γ2, or anti-Sds22 antibodies, as indicated, demonstrate that PPP1R11, PP1γ2, and Sds22 co-migrate.</p