milk ppak1 2 t423 402 ab 330220 cell signaling technology  (Cell Signaling Technology Inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: PPP1R12B inhibits cell proliferation by inducing G0/G1 phase arrest via PAK2/β-catenin axis in hepatocellular carcinoma

doi: 10.3389/fcell.2025.1621705

Figure Lengend Snippet: PAK2 plays a key role in PPP1R12B-mediated HCC proliferation suppression. (A) Schematic overview of the phosphoproteomic profiling strategy comparing PPP1R12B-overexpressing Huh7 cells with vector controls. The workflow includes protein extraction, tryptic digestion, phosphopeptide enrichment, LC-MS/MS analysis, and database search. (B) Protein interaction network of differentially phosphorylated proteins, with PPP1R12B positioned as a central node. The network was constructed using STRING with a confidence score threshold of 0.7. (C) Co-immunoprecipitation analysis demonstrating physical interaction between PPP1R12B and PAK2. Left: Flag-tagged PPP1R12B immunoprecipitated endogenous PAK2 in both Huh7 and HepG2 overexpression cells. Right: Reciprocal co-IP confirmed the interaction using PAK2 antibody for pulldown. (D) Immunofluorescence microscopy revealing subcellular co-localization of PPP1R12B (green) and PAK2 (red) in HCC cells. Nuclei were counterstained with DAPI (blue). Scale bars: 50 μm. (E) Functional rescue experiments showing that PAK2 knockdown (siPAK2) abrogated the proliferative effects of PPP1R12B modulation in CCK-8 assays.

Article Snippet: The primary antibodies used in Immunofluorescence were same as in Western blotting, plus PAK2 antibody (19979-1-AP, Proteintech).

Techniques: Plasmid Preparation, Protein Extraction, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Construct, Immunoprecipitation, Over Expression, Co-Immunoprecipitation Assay, Immunofluorescence, Microscopy, Functional Assay, Knockdown, CCK-8 Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: PPP1R12B inhibits cell proliferation by inducing G0/G1 phase arrest via PAK2/β-catenin axis in hepatocellular carcinoma

doi: 10.3389/fcell.2025.1621705

Figure Lengend Snippet: PPP1R12B suppresses proliferation via the PAK2/β-catenin/Cyclin D1 axis. (A) Confocal microscopy analysis demonstrating reduced β-catenin expression (green) following PAK2 (red) knockdown in Huh7 and HepG2 cells. Nuclei were counterstained with DAPI (blue). Scale bars: 50 μm. (B) PAK2-knockdown reduced total β-catenin, p-β-catenin (Ser675), and the expression of Cyclin D1 in CSQT-2 and HHL5 cells. (C) PPP1R12B overexpression decreased while knockdown increased the expression of PAK2, β-catenin, p-β-catenin (Ser675) and Cyclin D1 in HepG2 overexpression cells and CSQT-2 knockdown cells. (D,E) TOPFlash reporter assays measuring β-catenin transcriptional activity: (D) PAK2 knockdown significantly reduced β-catenin-mediated transcription (Huh7-P = 0.0003, HepG2-P = 0.0005); (E) PPP1R12B modulation correspondingly altered β-catenin activity (HepG2-P = 0.0411, CSQT-2-P = 0.0002, HHL5-P = 0.0037). (F) Subcellular fractionation analysis demonstrating PPP1R12B knockdown increased nuclear β-catenin accumulation. GAPDH and Lamin B1 served as compartment-specific controls. (G) CCK-8 assay revealed that palbociclib inhibited HCC cell proliferation and counteracted the proliferative changes induced by PPP1R12B modulation in HepG2 overexpression cells and CSQT-2 knockdown cells. The data were presented as mean ± SEM.

Article Snippet: The primary antibodies used in Immunofluorescence were same as in Western blotting, plus PAK2 antibody (19979-1-AP, Proteintech).

Techniques: Confocal Microscopy, Expressing, Knockdown, Over Expression, Activity Assay, Fractionation, CCK-8 Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: PPP1R12B inhibits cell proliferation by inducing G0/G1 phase arrest via PAK2/β-catenin axis in hepatocellular carcinoma

doi: 10.3389/fcell.2025.1621705

Figure Lengend Snippet: Clinical correlation between PPP1R12B and PAK2/β-catenin in HCC patients. (A) Transcriptomic analysis of PAK2 expression across multiple HCC cohorts ( GSE22058 , GSE36376 , GSE14520 , OEP000321) demonstrating significant upregulation in tumor tissues versus adjacent non-tumor controls ( GSE22058 -P = 0.0070, GSE36376 -P = 6.89 × 10 −26 , GSE14520 -P = 9.48 × 10 −39 , OEP000321-P = 4.38 × 10 −18 ). Boxplots represent median values with interquartile ranges. Data presented as log2-transformed TPM values. (B) Comparative analysis of CTNNB1 (β-catenin) mRNA levels showing consistent overexpression in HCC specimens across above datasets ( GSE22058 -P = 1.14 × 10 −8 , GSE36376 -P = 0.0406, GSE14520 -P = 4.77 × 10 −25 , OEP000321-P = 2.90 × 10 −17 ). Boxplots represent median values with interquartile ranges. Data presented as log2-transformed TPM values. (C) Spearman correlation analysis of TCGA-LIHC data revealing significant inverse relationships: PPP1R12B vs. PAK2: r = −0.2417, P = 0.0308; PPP1R12B vs. CTNNB1: r = −0.2489, p = 0.0260.

Article Snippet: The primary antibodies used in Immunofluorescence were same as in Western blotting, plus PAK2 antibody (19979-1-AP, Proteintech).

Techniques: Expressing, Transformation Assay, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: PPP1R12B inhibits cell proliferation by inducing G0/G1 phase arrest via PAK2/β-catenin axis in hepatocellular carcinoma

doi: 10.3389/fcell.2025.1621705

Figure Lengend Snippet: Schematic model. PPP1R12B suppresses HCC cell proliferation through the PAK2/β-catenin/Cyclin D1 axis. PPP1R12B could interact with PAK2 to suppress the expression and Ser675 phosphorylation of β-catenin, thereby inhibiting its nuclear translocation and the expression of its downstream target gene CCND1 , which regulated cell proliferation.

Article Snippet: The primary antibodies used in Immunofluorescence were same as in Western blotting, plus PAK2 antibody (19979-1-AP, Proteintech).

Techniques: Expressing, Phospho-proteomics, Translocation Assay