custom-made his 6 -trigger factor (tf)-ncx1 cyt and gst-pp1α  (GenScript corporation)

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: PP1c binds directly to NCX1. A, epitope mapping was performed by overlaying an array of immobilized overlapping 20-mer rat PP1-α (P62138) peptides with anti-PP1 (E-9) (sc-7482, top panel). Amino acids in bold constitute the core epitope, localized in the PP1 catalytic subunit (PP1c), and are relevant for anti-PP1c binding (n = 2). Immunodetection without anti-PP1c was used as a negative control (bottom panel). B, NCX1 and PP1c were analyzed in cytoplasmic and membrane fractions isolated from rat LV and neonatal cardiomyocytes using anti-NCX1 and anti-PP1c (in triplicate). Vinculin and epidermal growth factor receptor (EGFR) were used as controls for cytoplasmic and membrane fractions, respectively. C, rat LV lysates were subjected to immunoprecipitation (IP) using anti-NCX1 (in triplicate). Immunoprecipitates (right panels) and lysate (input; positive control for the immunoblot, left panels) were immunoblotted with NCX1, PP1c, and PLM antibodies. As a negative control, NCX1 antibody pre-incubated with a specific anti-NCX1 blocking peptide or non-relevant rabbit IgG was used. D, confocal images of in situ proximity ligation assay. NCX1-PP1c co-localization was analyzed in adult cardiomyocytes using anti-NCX1 and anti-PP1c (left panel, interaction indicated by green spots, see “Experimental Procedures” for details). Incubation without primary antibodies (middle panel) and pre-incubating anti-NCX1 with blocking peptide (right panel) were used as negative controls. Nuclei were stained with SYTOX orange. E, schematic figure of recombinant proteins used in this study. The cytosolic loop of NCX1 was purified, and His6 and trigger factor (TF) tags were added to the N terminus (His6-TF-NCX1cyt, top panel). Because of the size of the tags, His6-TF-NCX1cyt migrates as a 150-kDa protein. Commercially available PP1α was used in the initial analysis of the interaction (in F) after which rat PP1-α (P62138) with a GST tag (GST-PP1α) was generated (bottom panel) and used in the remainder of the study. F, recombinant proteins were subjected to immunoprecipitation using anti-NCX1 and anti-His6 (in triplicates). Immunoprecipitates (right panels) and proteins (input panels; positive control for the immunoblot) were immunoblotted with anti-NCX1 and anti-PP1c. As a negative control, non-relevant rabbit and mouse IgG were used. G, SPR analysis was performed by immobilizing recombinant His6-TF-NCX1cyt (ligand) on a CM5 chip and measuring the response when injecting a range of concentrations of GST-PP1α (analyte) (n = 3).

Article Snippet: The beads with bound peptides were then washed three times with PBS followed by incubation with 1 μg of GST-PP1α recombinant protein in 100 ml of IP-buffer supplemented with 1% BSA (805095, Bio-Rad) followed by gentle rotation for 2 h at 4 °C.

Techniques: Binding Assay, Immunodetection, Negative Control, Isolation, Immunoprecipitation, Positive Control, Western Blot, Incubation, Blocking Assay, In Situ, Proximity Ligation Assay, Staining, Recombinant, Purification, Generated

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Mapping of the PP1c interaction site in NCX1. A, schematic figure showing full-length NCX1-GFP and a series of GFP-NCX1 deletion mutants that were generated to map the PP1c interaction site in NCX1. The numbering of NCX1 includes the N-terminal amino acid signal peptide sequence. B, GST-PP1α together with NCX1 deletion variants were subjected to immunoprecipitation (IP) using anti-PP1c (in triplicates). Precipitates were analyzed with immunoblotting. The asterisks in the top panel indicate the NCX1 dimer (64) and missing GFP-NCX1(243–402) fragment. Noteworthy, the GFP-NCX1(243–402) fragment was not visible when we re-ran the samples on a second gel with good separation around 50 kDa and used a light chain-specific antibody (data not shown). The middle panel shows anti-PP1c-precipitated GST-PP1α. In the bottom panel the input lysates were immunoblotted to show the migration and corresponding molecular weight of NCX1-GFP and the deletion mutants. The asterisks in this panel indicate the NCX1 dimer, monomer, and migration of the GFP-NCX1(243–402) deletion fragment. C, identification of PP1 binding by overlaying GST-PP1α on 20-mer overlapping NCX1 peptides synthesized on membrane. Rat NCX1 protein (EDM02743) was used spanning amino acids 243–799. Binding was analyzed using anti-GST HRP (n = 2) top panel. Boxed areas show KVFF and KVLF respectively. Underlined amino acids in the first boxed area represent the RVXF -PP1-binding motif and a putative Φ1Φ2 motif, whereas amino acids in bold indicate the common sequence in the four peptide sequences. Incubation without GST-PP1α was used as a negative control (lower panel). D, one of the peptide sequences identified in C; 402PVSKVFFEQGTYQCLENCGT421, was synthesized with a glycine spacer and was overlaid with GST-PP1α (right panel) to determine the effect on PP1 binding when EN, putative Φ1Φ2 motif, was deleted (n = 2). The peptide sequence without GST-PP1α served as negative control (left panel).

Article Snippet: The beads with bound peptides were then washed three times with PBS followed by incubation with 1 μg of GST-PP1α recombinant protein in 100 ml of IP-buffer supplemented with 1% BSA (805095, Bio-Rad) followed by gentle rotation for 2 h at 4 °C.

Techniques: Generated, Sequencing, Immunoprecipitation, Western Blot, Migration, Molecular Weight, Binding Assay, Synthesized, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Confirmation of the PP1c interaction site in NCX1 by mutagenesis. NCX1(FL)-GFP, containing the KIFF motif (mouse sequence), together with either GFP-NCX1(I406A, F408A) representing KAFA mutant (A), GFP-NCX1(K405A, F407A) representing the AIAF mutant (B), or GFP-NCX1(F407P) representing the proline substitution KIPF and GFP-NCX1(Δ399–424) representing KIFF deletion (C) were generated and expressed in HEK293 cells after which they were used in immunoprecipitation (IP) experiments. The NCX1 antibody was used to precipitate NCX1 followed by immunoblotting with anti-PP1c for determination of PP1 association. D, rat LV lysates were subjected to immunoprecipitation using anti-NCX1, with and without the addition of 3 mm CaCl2. Immunoprecipitates (right panels) and lysate (input panels, positive controls for the immunoblot) were immunoblotted using relevant antibodies. E, peptide sequence 402PVSKVFFEQGTYQCLENCGT421 (identified in Fig. 3C) with KVFF motif was mutated to KAFA and AVAF and overlaid with GST-PP1α to determine the effect of mutations (n = 2). GST-PP1α binding was analyzed by immunodetection with anti-GST HRP. All experiments were run in triplicates (A–D).

Article Snippet: The beads with bound peptides were then washed three times with PBS followed by incubation with 1 μg of GST-PP1α recombinant protein in 100 ml of IP-buffer supplemented with 1% BSA (805095, Bio-Rad) followed by gentle rotation for 2 h at 4 °C.

Techniques: Mutagenesis, Sequencing, Generated, Immunoprecipitation, Western Blot, Binding Assay, Immunodetection

Journal: The Journal of Biological Chemistry

Article Title: Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman *

doi: 10.1074/jbc.M115.677898

Figure Lengend Snippet: Independence and isoform specificity of the NCX1-KIFF/KVFF-anchoring site. A, schematic figure of NCX1-biotinylated peptides used in pulldown and PP1 activity assays. B, pulldown assay with the biotinylated NCX1 peptides and GST-PP1α recombinant protein (in triplicates). PP1 binding was analyzed by immunoblotting with anti-PP1c (upper panel). Anti-biotin HRP was used to show the presence of biotinylated peptides. Incubation of the GST-PP1α recombinant protein with only the beads was used as negative control. C, effect of NCX1-PP1 interaction on PP1 activity was assessed. In each experiment, 1 unit of active PP1c was incubated with a range of recombinant His6-TF-NCX1cyt protein concentrations or (D) biotinylated NCX1 peptides for 20 min, after which the activity was determined. Inhibitor 2 (PP1 inhibitor) was used as control for the assay. The KIFF/KVFF-anchoring motif, a putative Φ1Φ2 motif (EN) and a conserved arginine motif (R), are shown in the alignment of human, rat, mouse NCX1 (E) and NCX1–3 isoforms (F) (black boxes indicate similar functional amino acids (DNA Star). G, pulldown assay with the biotinylated NCX1–3 peptides and GST-PP1α recombinant protein (in triplicates). PP1 binding was analyzed by immunoblotting with anti-PP1c (upper panel). Incubation of GST-PP1α recombinant protein with only the beads was used a negative control. GST-PP1α recombinant protein and biotinylated NCX peptides were used as positive controls for the immunoblot in B and G (input panels on left).

Article Snippet: The beads with bound peptides were then washed three times with PBS followed by incubation with 1 μg of GST-PP1α recombinant protein in 100 ml of IP-buffer supplemented with 1% BSA (805095, Bio-Rad) followed by gentle rotation for 2 h at 4 °C.

Techniques: Activity Assay, Recombinant, Binding Assay, Western Blot, Incubation, Negative Control, Functional Assay