Journal: Experimental and Therapeutic Medicine

Article Title: Small interfering RNA-mediated knockdown of KRT80 suppresses colorectal cancer proliferation

doi: 10.3892/etm.2020.9306

Figure Lengend Snippet: Knockdown of KRT80 expression decreases the expression levels of cell cycle-related proteins. SW480 cells were transfected with siRNA-KRT80 or siRNA-NC for 48 h. (A) Western blotting was used to analyze the expression levels of KRT80, PPP1CA, p21 and p27. (B) Semi-quantification of the western blotting data in part A. β-actin was used as the internal loading control for the western blotting analysis. Data are presented as the mean ± SD from 3 independent experimental repeats. * P<0.05, ** P<0.01 vs. siRNA-NC group. KRT80, keratin 80; PPP1CA, protein phosphatase 1 catalytic subunit α; p21, cyclin-dependent kinase inhibitor 1A; p27, cyclin-dependent kinase inhibitor 1B; siRNA, small interfering RNA; NC, negative control.

Article Snippet: The separated proteins were subsequently transferred to PVDF membranes (EMD Millipore) and blocked with 5% skimmed milk at room temperature for 1 h. The membranes were incubated with the following primary antibodies overnight at 4˚C: Anti-KRT80 (1:800; cat. no. 16835-1-AP; ProteinTech Group, Inc.), anti-protein phosphatase 1 catalytic subunit α (PPP1CA; 1:2,000; cat. no. 67070-1-Ig; ProteinTech Group, Inc.), anti-cyclin-dependent kinase inhibitor 1A (p21; 1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-cyclin-dependent kinase inhibitor 1B (p27; 1:1,000; cat. no. 3686; Cell Signaling Technology, Inc.) and anti-β-actin (1:2,500; cat. no. A5441; Sigma-Aldrich; Merck KGaA).

Techniques: Expressing, Transfection, Western Blot, Small Interfering RNA, Negative 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: 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: Molecular Biology of the Cell

Article Title: A Specific Form of Phospho Protein Phosphatase 2 Regulates Anaphase-promoting Complex/Cyclosome Association with Spindle Poles

doi: 10.1091/mbc.E09-07-0598

Figure Lengend Snippet: Depletion of the PPP2 subunits CA, R1A, or R2B leads to defective spindle formation and chromosome congression. (A) HeLa cells were transfected with siRNA targeting CA, R1A, or R2B for 48 h. The extent of gene knockdown was assessed by immunoblotting with antibodies directed against CA, R1A, R2B, and actin or GAPDH as loading controls. (B) Depletion of CA, R1A, or R2B leads to centrosome, mitotic spindle assembly, and chromosome congression defects. RNAi-treated cells in A were fixed and stained with Hoechst 33342, anti-α-tubulin, anti-pericentrin, and anti-centrin antibodies to visualize DNA, the mitotic spindle, the pericentriolar material, and the centrioles, respectively. Three groups of cell phenotypes were observed upon depletion of PPP2 subunits and are designated as groups 1 through 3. Group 1, has centrosome amplification and chromosome congression defects along with multipolar spindle formation. Group 2, contains chromosome cohesion defects and defective spindles. Group 3, exhibits defective spindle formation, spindle pole fragmentation, and a failure to congress chromosomes properly. Bar, 5 μm.

Article Snippet: The following antibodies were used: mouse anti-Cdc27 (Western blot), Eg5 (BD Transduction Laboratories, Lexington, KY), mouse anti-Cdh1 (Neomarkers, Fremont, CA), rabbit anti-Cdc20, cyclin D, cyclin B (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PPP1CA, PPP2CA, PPP2R1A, and PPP3CA (Upstate), rabbit anti-PPP2R2B (Bethyl Labs, Montgomery, TX), rabbit anti-PPP4C (Novus Biologicals, Littleton, CO), mouse anti-PPP5C (BD Transduction Laboratories), rabbit anti-PPP6C (Chemicon, Temecula, CA), rat anti-α-tubulin (Serotec), goat anti-GAPDH (Novus Biologicals), rabbit anti-pericentrin (Abnova), and rabbit anti-Emi1 (Zymed Laboratories, South San Francisco, CA).

Techniques: Transfection, Knockdown, Western Blot, Staining, Amplification