mouse anti pp1α  (Proteintech)

Journal: Cell Reports Methods

Article Title: Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale

doi: 10.1016/j.crmeth.2025.101084

Figure Lengend Snippet: Global phosphoproteome-wide in vitro phosphatase assays of PP1 and PP2A (A) Volcano plot highlighting identified phosphatase subunits in StrepHA-PPP1CA (right) and StrepHA-PPP2CA (left) purifications (marked in red) under native conditions. Dotted lines mark significance threshold; significantly enriched proteins marked in blue ( n = 3 biological replicates, FDR <0.05). Significantly enriched phosphatase subunits are annotated and highlighted as protein-interaction networks using information present in STRING DB. (B) Differentially regulated PPP1CA/PPP2CA sites, compared to either untreated control sample (left), or phosphatase- and okadaic acid-treated samples (OA, right). In vitro significantly regulated sites are highlighted in blue ( n = 3 biological replicates; FDR <0.05). (C and D) Motif and preference analyses of PPP1CA- and PPP2CA-sensitive sites. In motif analyses phosphatase-sensitive and OA-sensitive and insensitive sites are compared. Coloring corresponds to biochemical properties of residues. In preference analyses, the relative abundance of a given amino acid in a given position is indicated comparing phosphatase- and OA-sensitive (fold change of ≥3) to phosphatase-insensitive phosphosites. Colored lines next to amino acid names signify amino acid characteristics: green for hydrophobic, blue for basic, red for acidic, and gray for neutral amino acids. See also .

Article Snippet: Mouse monoclonal anti PPP1CA , Santa Cruz Biotech , Cat# sc-7482; RRID: AB_628177.

Techniques: In Vitro, Control

Journal: Cell Reports Methods

Article Title: Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale

doi: 10.1016/j.crmeth.2025.101084

Figure Lengend Snippet: Characterization of bona fide PP2A-PPP2R5E target sites (A) Complexome profiling of StrepHA-PPP2R5E purifications indicates enrichment of holocomplexes. (B) Volcano plot highlighting identified phosphatase subunits in StrepHA- PPP2R5E (left) and StrepHA-PPP1CA (right) purifications (marked in red) under native conditions. Dotted lines mark significance threshold, significantly enriched proteins marked in blue ( n = 3 biological replicates, FDR <0.05). Significantly enriched phosphatase subunits are annotated. (C) Significantly regulated phosphosites for PPP2R5E-PP2A complexes being OA sensitive ( n = 3 biological replicates). Significant sites are highlighted in blue (FDR <0.05). (D) Comparison of OBIPhA and in vivo analyses. Venn diagram highlights the overlap of identified phosphosites using the different experimental approaches. Bona fide PPP2R5E-PP2A target sites are defined as being regulated in vivo and in vitro (i.e., 194 sites on 168 proteins). (E) SLiMs in the bona fide PPP2R5E/B56ε-PP2A targets. (F) GO enrichment analysis using STRING DB highlighting biological processes of PPP2R5E-PP2A target proteins. (G) Targeted, phosphosite-specific MS analysis (PRM) for DDX3X Ser90 and Ser609. Shown is the quantification of three replicates (black dots). ∗ p < 0.05; ∗∗ p < 0.01; t test. (H) IF of CAPRIN1 and G3BP1. Shown are exemplary images of n = 3 biological replicates. Nuclei are stained in blue. Scale bar, 10 μm. Box plots show quantifications of 12 images of n = 3 biological replicates, boxes are drawn according to ggplot2 standard settings. White dots indicate average stress granule number/mean volume per cell and image. ∗ p < 0.05; ∗∗ p < 0.01; t test. See also .

Article Snippet: Mouse monoclonal anti PPP1CA , Santa Cruz Biotech , Cat# sc-7482; RRID: AB_628177.

Techniques: Comparison, In Vivo, In Vitro, Phospho-proteomics, Staining

Journal: bioRxiv

Article Title: C12ORF57: a novel principal regulator of synaptic AMPA currents and excitatory neuronal homeostasis

doi: 10.1101/2025.01.08.632037

Figure Lengend Snippet: a) Western blot showing co-immunoprecipitation of CAMK4 with C12ORF57 from Grcc10+/+ whole brain lysate (left) with Grcc10−/− brain negative control (right) showing no GRCC10 expression or CAMK4 pulldown b) Representative CAMK4 and GAPDH western blot bands from brain whole cell lysates of Grcc10 +/+ and Grcc10−/− mice c) Mean relative CAMK4 western blot band intensity normalized to WT (p=.34, N=6 for each genotype Welch’s T-Test) Error bars represent SEM. d) Representative CAMK4, pCAMK4 and lamin (control) bands on western blot from nuclear fraction of Grcc10 WT and KO whole brain lysates e) Relative CAMK4, pCAMK4and Lamin band intensity normalized to Grcc10 WT levels. (p=.001, N=6 for each genotype, 2-way ANOVA with Sidak’s multiple comparison test). f) Representative CAMK4, pCAMK4 and GAPDH (control) bands on western blot from cytoplasmic fractions of whole brain g) Relative CAMK4 pCAMK4 and Lamin band intensity normalized to Grcc10 WT levels (p=.016, N=6 for each genotype, 2-way ANOVA, Sidak’s multiple comparison test) in KO compared to WT. h) Schematic of WT CAMK4 (top) with kinase domain (blue), calmodulin binding domain (purple) and autoinhibitory/regulatory domain (yellow). Construct Δ(322-341) which lacks the autoinhibitory domain (NAI, middle) and construct Δ(305-321) which lacks the calmodulin binding domain (NCB, bottom) i) Representative western blot of co-IP of CAMK4 constructs with C12ORF57-FLAG j ) Relative CAMK4 band intensity between WT, NCB (p=.24, N=8 replicates for each construct, ANOVA) and NAI (p<.0001, N=8 replicates for each construct, ANOVA) CAMK4 constructs.. k ) Relative luminescence from in vitro CAMK4 kinase activity assay for C12ORF57, CAMK4, CAMK4+C12ORF57, CAMK4+PP1CA, CAMK4+PP1CA+C12ORF57, no CAMK4, and no ATP (N=3 per condition, ANOVA). l ) Left: Graph of cumulative frequency (x-axis) of mEPSC against amplitude (pA) in DIV 17 pyramidal neurons including CAMK4 transfected control (green) KO (pink) WT (black), and C12ORF57 transfected (purple). The mean curve of each group is bolded. Right: Mean amplitude of WT, KO, CAMK4 transfected (KO+CAMK4) and C12OR57 transfected (KO+C12ORF57) (p<.0001, N=6 for each group, ANOVA). For all graphs, error bars represent SEM. Statistical differences are indicated in the figures using the following symbols * P ≤ .05, ** P ≤ .01, *** P ≤ .001, **** P < .0001.

Article Snippet: CAMK4, C12ORF57, and purified recombinant PP1CA (Origene, TA808819) were mixed in equimolar amounts with 25 µM ATP and reaction luminescence was measured on a Tecan Spark for an integration time of 1 sec.

Techniques: Western Blot, Immunoprecipitation, Negative Control, Expressing, Control, Comparison, Binding Assay, Construct, Co-Immunoprecipitation Assay, In Vitro, Kinase Assay, Transfection