liprin β1  (Santa Cruz Biotechnology)

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: Identification of the target of sTN58 aptamer on TNBC cell surface. (A) Schematic representation of biotin-sTN58-mediated affinity purification. Membrane-protein fraction from Cis-Pt-R cells were subjected to a preclearing step to remove non-specific components prior to the sTN58-mediated precipitation. The colloidal Blue-stained SDS-PAGE (10 %) displayed is utilized for the analysis of target purification mediated by the sTN58 aptamer. The molecular weights of protein markers are reported. Lane 1, molecular markers; lane 2, membrane extracts; lane 3, 15 μg aliquot of unbound proteins from SCR-mediated purification; lane 4, proteins captured with sTN58. Red boxes indicate the regions excised for MS analyses. (B) Comparison of transcript expression values of best candidates in different BC cell lines. The normalized transcript expression values (nTPM), according to HPA, are reported relative to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. Box indicates the 5 candidates chosen for experimental validation. (C) Immunoblot analysis of EphA2, CD44, integrin β1, myoferlin, liprin β1 and ZO-1, and of the housekeeping proteins α-tubulin and vinculin. The molecular weights of protein markers are reported. Black dashed lines delineate the boundary between non-contiguous lanes of the same gel. (D) The histogram shows the relative fold-change in expression levels of the indicated proteins compared to the housekeeping protein α-tubulin or vinculin, normalized to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. (E) Binding affinity (1/Kd) of TN58 aptamer to the indicated cell lines expressed relative to MDA-MB-231 target cells. Dose response curves and binding affinity calculations for MDA-MB-231 and Cis-Pt-R and Dox-R chemoresistant cells, as well as non-TNBC BT-474, MCF-7 and A431 cells were previously reported [ , ]. The dose response curve used for Kd calculation in relation to BT-549 is shown in . "NB", no binding.

Article Snippet: Filters were incubated overnight at 4 °C with the following primary antibodies: CD44 (lysates from human cell lines), Zonula occludens-1 (ZO-1, D7D12), platelet-derived growth factor receptor β (PDGFRβ, 28E1), phospho-44/42 MAPK (extracellular signal-regulated kinase 1/2, ERK1/2, D13.14.4E, indicated as p-ERK1/2), phospho-Akt (Ser473, indicated as p-Akt), Akt, Met (25H2), vimentin (D21H3), E-Cadherin (24E10), vinculin (E1E9V), α-tubulin (DM1A) (Cell Signaling Technology Inc., Danvers, MA, USA); CD44 (lysates from murine 4T1 cells, ab157107), integrin β1 (ITGB1, ab179471) (Abcam, Cambridge, UK); liprin β1, Ephrin Type-A Receptor 2 (EphA2), ERK1 (C-16) (Santa Cruz Biotechnology, Santa Cruz, CA); myoferlin (MYOF, HPA014245, Sigma-Aldrich) and programmed cell death-ligand 1 (PD-L1)/CD274 (Proteintech Group, Inc.).

Techniques: Affinity Purification, Membrane, Staining, SDS Page, Purification, Comparison, Expressing, Biomarker Discovery, Western Blot, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liprin-α1 liquid-liquid phase separation

doi: 10.1016/j.jbc.2025.110349

Figure Lengend Snippet: Identification of SLiM4 as the functional PPP2R5D-interaction motif within liprin-α1. A , eight consensus LxxIxE short linear motifs (SLiMs) were identified in liprin-α1, potentially mediating the interaction with PPP2R5D. The amino acid sequence and residue numbers are listed for each motif, as well as their presence within ordered versus disordered parts of the protein. B , FLAG-tagged full-length liprin-α1 (WT) or N-terminal liprin-α1 deletions lacking the first 30 (Δ1-30), 152 (Δ1-152) or 180 amino acids (Δ1-180) were either cotransfected with GFP-tagged PPP2R5D or GFP alone in HEK293 cells. Following GFP-trapping, the presence of FLAG-tagged proteins in the GFP pull downs was verified by immunoblotting. C , HA-tagged full-length liprin-α1 (WT), indicated HA-tagged liprin-α1 SLiM mutants, or HA alone (control) were expressed in HEK293 cells, and binding of endogenous PPP2R5D was assessed in anti-HA immunoprecipitates. D , GFP-tagged full-length liprin-α1 (WT), GFP-tagged liprin-α1 SLiM1, SLiM4, or SLiM1+4 mutants, or GFP alone (control) were expressed in HEK293 cells, and binding of endogenous PPP2R5D was assessed in GFP traps. E , quantifications of the PPP2R5D/liprin-α1 ratios in each condition are shown (unpaired t test, N = 3 technical replicates, ns: not significant, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001), clearly demonstrating the functionality of SLiM4. HA, hemagglutinin.

Article Snippet: Following SDS-PAGE and immunoblotting as in ( , ), the pull-downs were counterstained with anti-GFP (Cell Signaling Technologies, # 2555S), anti-FLAG antibodies (Sigma-Aldrich, #F1804), anti-PPP2R5D (Abcam, # ab188323) or anti-liprin-β1 antibody (Atlas Antibodies, # HPA001924).

Techniques: Functional Assay, Sequencing, Residue, Western Blot, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liprin-α1 liquid-liquid phase separation

doi: 10.1016/j.jbc.2025.110349

Figure Lengend Snippet: Liprin-α1 SLiM4 mutant significantly increased GFP-liprin-α1 liquid-liquid phase separation. A , representative live confocal images of WT HEK293 cells transfected with GFP-liprin-α1 (WT), GFP-liprin-α1 SLiM1 mutant (SLiM1), GFP-liprin-α1 SLiM4 mutant (SLiM4), or GFP-liprin-α1 SLiM1, and 4 mutant (SLiM1+4). The scale bar represents 20 μm. B , quantification of the percentage of WT HEK293 cells with droplets. Shown are mean ± SD as well as individual data points from three technical replicates. Significance was determined by one-way ANOVA with Dunnett's multiple comparison post hoc test (ns: not significant, ∗∗∗ p ≤ 0.001). C , an example of time-lapse confocal images of WT HEK293 cells transfected with SLiM4. Droplets subject to fluorescence recovery after photobleaching (FRAP). One hundred twenty frames acquired with 0.5 s between each frame. Bleaching occurred at frame 2 (t = 0 s). Images show the droplet directly before and after bleaching as well as the fluorescence recovery over time. D , normalized percentage of FRAP plotted over time. N = 30 droplets/3 technical replicates. Error bars indicate mean ± SD. SLiM, short linear motif.

Article Snippet: Following SDS-PAGE and immunoblotting as in ( , ), the pull-downs were counterstained with anti-GFP (Cell Signaling Technologies, # 2555S), anti-FLAG antibodies (Sigma-Aldrich, #F1804), anti-PPP2R5D (Abcam, # ab188323) or anti-liprin-β1 antibody (Atlas Antibodies, # HPA001924).

Techniques: Mutagenesis, Transfection, Comparison, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liprin-α1 liquid-liquid phase separation

doi: 10.1016/j.jbc.2025.110349

Figure Lengend Snippet: Mimicking liprin-α1 phosphorylation increased its liquid-liquid phase separation. A , MS-based targeted phospho analysis of GFP-trapped liprin-α1 WT and SLiM4 mutant from transfected HEK293 cells (n = 1). Phosphorylated peptide abundances were normalized according to the protein abundance of liprin-α1. The position of the phosphorylated amino acid(s) in the liprin-α1 protein is indicated. B , representative confocal images of live WT HEK293 cells transfected with GFP-liprin-α1 phospho-mimetic mutants (S150E, S458E, S666E, S668E, S666/668E, S708E, S763E, or S1171/1172E). The scale bar indicates 20 μm. C , quantification of the percentage of WT HEK293 cells transfected with phospho-mimetic GFP-liprin-α1 mutants with droplets. Shown are mean ± SD as well as individual data points from three technical replicates. Significance was determined by one-way ANOVA with Dunnett's multiple comparison post hoc test (not significant unless otherwise noted, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01). D , alignment of four liprin-α isoforms around S763 of liprin-α1. E , representative immunoblot of four technical replicates showing GFP or pS763 after GFP trapping using WT HEK293 cells transfected with GFP-liprin-α1 WT or SLiM4 mutant. F , quantification of the ratio of pS763 over total GFP band normalized to the WT condition. Shown are mean ± SD as well as individual data points from four technical replicates. Significance was determined by an unpaired t test (∗ p ≤ 0.05). MS, mass spectrometry; SLiM, short linear motif.

Article Snippet: Following SDS-PAGE and immunoblotting as in ( , ), the pull-downs were counterstained with anti-GFP (Cell Signaling Technologies, # 2555S), anti-FLAG antibodies (Sigma-Aldrich, #F1804), anti-PPP2R5D (Abcam, # ab188323) or anti-liprin-β1 antibody (Atlas Antibodies, # HPA001924).

Techniques: Phospho-proteomics, Mutagenesis, Transfection, Quantitative Proteomics, Comparison, Western Blot, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liprin-α1 liquid-liquid phase separation

doi: 10.1016/j.jbc.2025.110349

Figure Lengend Snippet: Increased liprin-α1 liquid-liquid phase separation in PPP2R5D knockout (KO) HEK293 cells and human de novo mutation E420K homozygous HEK293 cells. A , immunohistochemistry validation of PPP2R5D KO HEK293 cells. B , immunoblot of PPP2R5D KO and WT HEK293 cells showing PPP2R5D protein was successfully eliminated. β-actin was probed as an internal control. C , representative confocal images of WT, PPP2R5D KO, or E420K homozygous HEK293 cells transfected with GFP-liprin-α1. The scale bar indicates 20 μm. D , quantification of the percentage of cells containing droplets. Shown are mean ± SD as well as individual data points from three technical replicates. Significance was determined by one-way ANOVA with Dunnett's multiple comparison post hoc test (∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). E – G , normalized mean FRAP over time for GFP-liprin-α1 droplets in ( E ) WT, ( F ) PPP2R5D KO, or ( G ) E420K homozygous HEK293 cells. N = 30 droplets/3 technical replicates per cell line. Error bars indicate mean ± SD. H and J , representative immunoblot of GFP or pS763 after GFP trapping using WT, PPP2R5D KO, or E420K homozygous HEK293 cells transfected with GFP-liprin-α1. I and K , quantification of the ratio of pS763 over total GFP band normalized to the WT condition. Shown are mean ± SD as well as individual data points from three to five technical replicates. Significance was determined by unpaired t test (∗ p ≤ 0.05, ∗∗∗ p ≤ 0.01). FRAP, fluorescence recovery after photobleaching.

Article Snippet: Following SDS-PAGE and immunoblotting as in ( , ), the pull-downs were counterstained with anti-GFP (Cell Signaling Technologies, # 2555S), anti-FLAG antibodies (Sigma-Aldrich, #F1804), anti-PPP2R5D (Abcam, # ab188323) or anti-liprin-β1 antibody (Atlas Antibodies, # HPA001924).

Techniques: Knock-Out, Mutagenesis, Immunohistochemistry, Biomarker Discovery, Western Blot, Control, Transfection, Comparison, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liprin-α1 liquid-liquid phase separation

doi: 10.1016/j.jbc.2025.110349

Figure Lengend Snippet: Domain analysis of liprin-α1 in its liquid-liquid phase separation. A , schematic of liprin-α1 domains showing the design of constructs containing different liprin-α1 domains. Constructs include deletion of the intrinsically disordered region (ΔIDR), deletion of all three SAM domains (ΔSAMs), the SAM domains alone (SAMs), and the N terminus only (1-344, N-term). Constructs are tagged with GFP or mScarlet as noted. B and D , example live confocal images of WT HEK293 cells ( B ) or PPP2R5D KO HEK293 cells ( D ) transfected with GFP-ΔIDR, GFP-ΔSAMs, mScarlet-SAMs, or mScarlet-N-term. The scale bar indicates 20 μm. C and E , quantification of the percentage of ( C ) WT HEK293 cells or ( E ) PPP2R5D KO HEK293 cells transfected with various liprin-α1 domain mutants containing droplets. Shown are mean ± SD as well as individual data points from three technical replicates. Significance was determined by one-way ANOVA with Dunnett's multiple comparison post hoc test (∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001). IDR, intrinsically disordered region; SAM, sterile alpha helical motif.

Article Snippet: Following SDS-PAGE and immunoblotting as in ( , ), the pull-downs were counterstained with anti-GFP (Cell Signaling Technologies, # 2555S), anti-FLAG antibodies (Sigma-Aldrich, #F1804), anti-PPP2R5D (Abcam, # ab188323) or anti-liprin-β1 antibody (Atlas Antibodies, # HPA001924).

Techniques: Construct, Transfection, Comparison, Sterility

Journal: The Journal of Biological Chemistry

Article Title: The phosphatase activity of the PPP2R5D-PP2A holoenzyme modulates liprin-α1 liquid-liquid phase separation

doi: 10.1016/j.jbc.2025.110349

Figure Lengend Snippet: Inhibition of liprin-α1 binding to liprin-β1 increased liprin-α1 liquid-liquid phase separation. A , example confocal images of live WT HEK293 cells transfected with GFP-liprin-α1 WT or GFP-liprin-α1 with an E942A point mutation to inhibit liprin-β1 binding. The scale bar indicates 20 μm. B , quantification of the percentage of transfected WT HEK293 cells with droplets. Shown are mean ± SD as well as individual data points from three technical replicates. Significance was determined by unpaired t test (∗ p ≤ 0.05). C , example immunoblots of GFP, PPP2R5D, and liprin-β1 on HEK293 cell lysates (input (IN), left ) or immunoprecipitated elutes (GFP-trapping (IP), right ). WT HEK293 cells were transfected with GFP-liprin-α1 WT or mutants (E942A, SLiM4, or S763E). KO and E420K homozygous HEK293 cells were transfected with GFP-liprin-α1 WT. D , quantification of the percentage of co-IP of liprin-β1 with GFP-liprin-α1 normalized to GFP-liprin-α1 transfected WT HEK293 cells. Shown are the mean ± SD and individual data points from four technical replicates. Significance was determined by one-way ANOVA with Dunnett's multiple comparison post hoc test (∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). E , quantification of the percentage of co-IP of PPP2R5D with GFP-liprin-α1 normalized to GFP-liprin-α1 transfected WT HEK293 cells. Shown are mean ± SD as well as individual data points from four or five technical replicates. Significance was determined by one-way ANOVA with Dunnett's multiple comparison post hoc test (ns: not significant, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001). co-IP, co-immunoprecipitation; SLiM, short linear motif.

Article Snippet: Following SDS-PAGE and immunoblotting as in ( , ), the pull-downs were counterstained with anti-GFP (Cell Signaling Technologies, # 2555S), anti-FLAG antibodies (Sigma-Aldrich, #F1804), anti-PPP2R5D (Abcam, # ab188323) or anti-liprin-β1 antibody (Atlas Antibodies, # HPA001924).

Techniques: Inhibition, Binding Assay, Transfection, Mutagenesis, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison