PMC

SPATA 18 interacts with 14-3-3 and B-raf. (A) HEK293 cells were transfected with pcDNA3.1 His-SPATA18 and pcDNA5.1 Myc-14-3-3 as described in materials and methods. The cell extracts were prepared 48 h after transfection and were subjected to Immunoprecipitation with the His-tag antibody and a control antibody. Immunoblotting of His-SPATA18 immuoprecipitates showed that Myc-14-3-3 (31 kDa) co-precipitate with His-SPATA18 (∼75 kDa). (B) HEK-293 cells were transfected with pcDNA3.1His-SPATA18 plasmid. The His-SPATA18 protein was immunoprecipitated with His-tag antibody and the immunoprecipation eluate was analyzed with anti-His and B-raf antibodies. B-raf (∼85 kDa) co-precipitated with His-SPATA18.

PP1 and PP2A bound to 14-3-3 are catalytically active. The TEV eluate isolated after first step of TAP purification was checked for the phosphatase activity of PP 1 and PP 2A as described in materials and methods. For protein phosphatase activity measurements, Phosphorylase a, a common substrate for both PP1 and PP2A was used. PP2A activity could be inhibited with 5 nM okadaic acid. The activity of both PP2A and PP1 could be inhibited to basal levels with 1 µM okadaic acid. (B) Protein gel blot analysis of the TEV eluate showed that both PP1 and PP2A interact with 14-3-3 in testis.

Tandem affinity purification. (A) A Schematic representation of the two steps of TAP-14-3-3 purification. The two-step procedure decreases the non-specific interactions. The purified proteins can be identified by protein gel blot analysis and/or mass spectrometry. (B) TAP-14-3-3 isolation was performed as described in the materials and methods. Detection of TAP-14-3-3 and endogenous 14-3-3 levels at each step of purification by protein gel blot analysis using an antibody against 14-3-3. (C) The EGTA eluate containing the 14-3-3 binding proteins was concentrated after TAP. The proteins were separated on a 12% SDS-PAGE stained with coomassie blue and the gel was used for the LC-MS/MS analysis.

PP1γ2 and PGK2 are in an endogenous complex with 14-3-3. Total testis lysate and the EGTA eluate after TAP were resolved by SDS-PAGE and transferred on a PVDF membrane followed by immunoblot analysis with (A) PP1γ2 and (B) PGK2 antiserum. (C) The TAP-14-3-3 complexes were captured on IgG beads. The IgG beads were washed with wash buffer and 14-3-3 complexes were eluated after TEV clevage. The TEV eluate was incubated with purified IgG (as a control) or PP1γ2 antibody to immunoprecipitate PP1γ2. Immunoblotting analysis of the control and PP1γ2 immunoprecipation eluate showed that both 14-3-3 and PGK2 can be co-precipitated specifically with PP1γ2 from the TEV eluate. (D–F) 14-3-3 complexes from the TAP transgenic mice testis were purified by the first step of TAP purification on IgG beads. The 14-3-3 complexes were eluated by TEV cleavage. The TEV eluate was incubated with microcystin beads to isolate PP1 complexes. The microcystin eluate was analyzed by protein gel blotting with PP1γ2, 14-3-3 and PGK2 antibodies.

TAP 14-3-3 transgene construct plan and TAP 14-3-3 expression analysis in testis. (A) A Diagrammatic representation of the TAP 14-3-3 cDNA construct. (B) The TAP tag consisted of a protein A tag and a calmodulin binding peptide (CBP) separated by the TEV cleavage site sequence. The TAP tag was fused to 14-3-3ζ, cDNA at its N-terminal end. (C) Protein gel blot analysis of 14-3-3 expression in testis. Both the wild type and transgenic mice testes extracts showed endogenous 14-3-3 protein migrating at 30 kDa whereas TAP 14-3-3 (50 kDa) could be detected only in the testis extracts of transgenic mice. (D) Immunohistochemical analysis of the TAP 14-3-3 transgenic mice testes showed that the TAP 14-3-3 is expressed in both the seminiferous tubules and the interstitial compartment of the testis. Testis sections from the wild type mice stained with rabbit IgG showed no fluorescence.