Journal:

Article Title: NHERF2 Specifically Interacts with LPA 2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-?3 Activation

doi: 10.1128/MCB.24.11.5069-5079.2004

Figure Lengend Snippet: NHERF2 couples LPA2 to PLC-β3 specifically. (A) LPA2 forms a molecular complex with PLC-β3 in a NHERF2-dependent manner. The NHERF2 constructs (NHERF2, NHERF2 WT; NHERF2 ΔPDZ2, the second PDZ domain-deleted form) were cotransfected in combination with either Flag-PLC-β3 (left) or Flag-PLC-β1 (right) as indicated. After 2 days, COS-7 cells were washed and incubated with PBS containing 0.5 mM DSP (Pierce), which is a cell-permeable cross-linker, for 30 min. After the residual DSP was blocked with PBS containing 50 mM Tris buffer, the cleared lysates were subjected to a pull-down assay using GST-LPA2-CT immobilized onto GSH beads. After a washing step, the resulting precipitates were subjected to SDS-PAGE and then analyzed by Western blot analysis with anti-Flag antibody (top) and anti-NHERF2 antibody (middle) or by Ponceau S staining (bottom) as indicated. The results are representative of three independent experiments. (B) Specific coimmunoprecipitation of PLC-β3 and not of PLC-β1 with Flag LPA2. HeLa cells (at a density of 6 × 106 cells/150-mm dish) were infected with either the recombinant Flag-LPA2 adenovirus or control empty virus at an MOI of 10. At 24 h postinfection, the infected cells were incubated with PBS containing 0.5 mM DSP for 30 min. After the cell lysates were blocked with 50 mM Tris buffer, they were immunoprecipitated with anti-Flag antibody. After a washing step, the resulting precipitates were analyzed with the specific antibodies as indicated. (C) Gene silencing of PLC-β isoforms with specific siRNAs. HeLa cells were transfected with siRNAs directed against PLC-β1 or PLC-β3, along with control siRNA (Luciferase), as described in Materials and Methods. After 3 days, the HeLa cells were lysed and then analyzed by Western blot analysis with specific antibodies, as indicated. (D) PLC-β3 is functionally coupled to LPA2. At 24 h after siRNA transfection, the cells were split at a density of 2 × 105 cells/35-mm well and infected with recombinant adenovirus expressing Flag-LPA2 for 4 h. At 12 h after viral infection, the infected cells were labeled with 1 μCi of [3H]inositol/ml for 12 h and treated with either 0.1% BSA only (−) or 1 μM LPA-BSA conjugates (+). The generation of [3H]IPs was analyzed as described in Materials and Methods. The results are presented as means ± the SE of three experiments performed in duplicate.

Article Snippet: In addition, the specific antibodies to PLC-β1, PLC-β3, and phosphorylated extracellular signal-regulated kinase (ERK) were obtained from the Santa Cruz Co., and the anti-COX-2 antibody was acquired from Caymen Chemicals (Ann Arbor, Mich.).

Techniques: Construct, Incubation, Pull Down Assay, SDS Page, Western Blot, Staining, Infection, Recombinant, Immunoprecipitation, Transfection, Luciferase, Expressing, Labeling

Journal:

Article Title: NHERF2 Specifically Interacts with LPA 2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-?3 Activation

doi: 10.1128/MCB.24.11.5069-5079.2004

Figure Lengend Snippet: Schematic view of the NHERF2-dependent regulation of LPA2-mediated PLC-β3 signaling. Prior to agonist stimulation, both LPA2 and PLC-β3 directly interact with NHERF2, which is localized to the plasma membrane. NHERF2, which is multimerized by a PDZ-PDZ interaction, clusters LPA2 and PLC-β3 in close proximity, thereby creating spatially compact signaling complexes beneath the plasma membrane. Consequently, the LPA2-NHERF2-PLC-β3 complex enables LPA2 to transduce its signal to PLC-β3 with efficiency and specificity.

Article Snippet: In addition, the specific antibodies to PLC-β1, PLC-β3, and phosphorylated extracellular signal-regulated kinase (ERK) were obtained from the Santa Cruz Co., and the anti-COX-2 antibody was acquired from Caymen Chemicals (Ann Arbor, Mich.).

Techniques: Transduction

Journal: The Journal of Experimental Medicine

Article Title: Epstein-Barr Virus Latent Membrane Protein 2a (Lmp2a) Employs the Slp-65 Signaling Module

doi:

Figure Lengend Snippet: Block of PLC-γ2 phosphorylation in LCLs. Anti–PLC-γ2 immunoprecipitates from unstimulated and BCR-stimulated LCLs (lanes 1–6) or Ramos B cells (lanes 7 and 8) were analyzed by immunoblotting with antibodies to pTyr (top panel), SLP-65 (middle panel), or PLC-γ2 (bottom panel). Relative molecular masses of marker proteins are indicated on the left in kD.

Article Snippet: After lysis of the unstimulated or stimulated B cells in 1% Nonidet P-40 buffer , immunoprecipitations were performed using agarose-coupled mouse monoclonal antibodies to phosphotyrosine (pTyr; 4G10; UBI), or with protein G-adsorbed mouse monoclonal antibodies to human SLP-65/BLNK (Babco), or with rabbit antibodies specific for the following BCR effector proteins: PLC-γ2 (Q20), CrkL, and C3G (Santa Cruz Biotechnology, Inc.); and Casitas B lineage lymphoma (Cbl; Transduction Laboratories/Becton Dickinson).

Techniques: Blocking Assay, Phospho-proteomics, Western Blot, Marker

Journal: Journal of Inflammation Research

Article Title: Significance of Immunogenic Cell Death-Related Prognostic Gene Signature in Cervical Cancer Prognosis and Anti-Tumor Immunity

doi: 10.2147/JIR.S410140

Figure Lengend Snippet: Expression of the PDIA3 gene in cervical cancer (CC) and its prognostic value. ( A ) The cell types of IPGs distribution in the single-cell analysis of the GSE168652 database were described. ( B ) UMAP visualization of PDIA3 in major cell subpopulations of cervical cancer. ( C ) The expression levels of HPV16- and HPV18-positive groups were compared with normal groups in the GSE6791 database (* p < 0.05, ** p < 0.01, and *** p < 0.001). ( D ) Cox survival analysis showing hazard ratios for different IPGs. ( E ) Differences in PDIA3 expression between normal and cancer tissue samples (* p < 0.05). ( F and G ) Kaplan–Meier curves showing differences in overall survival ( F ) and progression-free survival ( G ) between PDIA3 high and low expression groups; ( H – J ) PDIA3 protein expression in normal cervical tissue samples and CC tissue samples. Normal cervical tissue ( H ), cervical squamous cancer ( I ), and cervical adenocarcinoma tissue ( J ).

Article Snippet: PDIA3 antibody (Proteintech, Wuhan, China), GAPDH antibody (CST, Shanghai, China), goat anti-rabbit IgG antibody secondary antibody (Zsbio, Beijing, China), and goat anti-mouse IgG H&L/Cy3 (Beyotime, Shanghai, China).

Techniques: Expressing, Single-cell Analysis

Journal: Journal of Inflammation Research

Article Title: Significance of Immunogenic Cell Death-Related Prognostic Gene Signature in Cervical Cancer Prognosis and Anti-Tumor Immunity

doi: 10.2147/JIR.S410140

Figure Lengend Snippet: PDIA3 is highly expressed in cervical cancer (CC) tissues and cell lines. ( A and B ) Western blot analysis reveals a high expression of PDIA3 in CC cell lines. ( C ) Distribution of PDIA3 in CC cell lines. ( D ) Immunohistochemistry reveals PDIA3 expression in CC tissues, with normal cervical tissue serving as a positive control (200×). ( E ) IPGs mRNA expression relative to GAPDH in cervical cancer samples comparing (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: PDIA3 antibody (Proteintech, Wuhan, China), GAPDH antibody (CST, Shanghai, China), goat anti-rabbit IgG antibody secondary antibody (Zsbio, Beijing, China), and goat anti-mouse IgG H&L/Cy3 (Beyotime, Shanghai, China).

Techniques: Western Blot, Expressing, Immunohistochemistry, Positive Control

Journal: Molecular Cancer

Article Title: Integrative multi-omics analysis reveals the LncRNA 60967.1–PLCD4–ATRA axis as a key regulator of colorectal cancer progression and immune response

doi: 10.1186/s12943-025-02359-x

Figure Lengend Snippet: Experimental validation of mRNA, lncRNA, and metabolite levels. ( A ) Expression of lncRNA 60967.1 in CRC cell lines (DLD1, HCT8, SW480) relative to the normal colon cell line (NCM460), measured by qRT-PCR. ( B ) qRT-PCR validation of lncRNA 60967.1 expression in 12 paired CRC tumor and normal tissues. ( C ) qRT-PCR analysis of PLCD4 mRNA levels in the three CRC cell lines (DLD1, HCT8, SW480) and the normal colon cell line. ( D ) qRT-PCR validation of PLCD4 mRNA levels in 12 paired CRC tumor and normal tissues. ( E-F ) Western blot analysis of PLCD4 protein expression in the three CRC cell lines (DLD1, HCT8, SW480) and the normal colon cell line (NCM460) ( E ) and 12 paired tumor (T#) and normal (N#) tissues ( F ). ( G ) IHC analysis of PLCD4 protein expression in the three CRC cell lines (DLD1, HCT8, SW480) and the normal colon cell line (NCM460). ( H ) ELISA-based quantification of ATRA levels in culture supernatants from the three CRC cell lines (DLD1, HCT8, SW480) and the normal colon cell line (NCM460). *** P < 0.01 (t test)

Article Snippet: To capture the PLCD4-RNA complex, human PLCD4 antibody (30133-1-AP, Proteintech) and IgG antibody were incubated with Protein A + G beads overnight at 4 °C.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer

Article Title: Integrative multi-omics analysis reveals the LncRNA 60967.1–PLCD4–ATRA axis as a key regulator of colorectal cancer progression and immune response

doi: 10.1186/s12943-025-02359-x

Figure Lengend Snippet: Molecular and functional effects of lncRNA 60967.1 on PLCD4 expression, ATRA levels, and CRC cell proliferation. ( A ) Fluorescence in situ hybridization (FISH) showing the localization of lncRNA 60967.1 and PLCD4 mRNA in NCM460 cells. ( B ) RIP assay followed by qRT-PCR analysis of lncRNA 60967.1 in immunoprecipitated (IP) and IgG control groups. ( C ) RIP assay followed by Western blot analysis of PLCD4 in the input, immunoprecipitated (IP), and IgG control groups. ( D ) qRT-PCR analysis of PLCD4 mRNA in CRC cells (DLD1, HCT8, SW480) transfected with GFP control or lncRNA 60967.1 vector. ( E ) Western blot analysis of PLCD4 protein expression in CRC cells (DLD1, HCT8, SW480) with GFP or lncRNA 60967.1 vector. ( F ) ELISA measurement of ATRA levels in CRC cells (DLD1, HCT8, SW480) with GFP or lncRNA 60967.1 vector. ( G-I ) CCK-8 assay for CRC cell proliferation (DLD1, HCT8, SW480) transfected with lncRNA 60967.1 vector, followed by IFN-γ stimulation. ( J ) qRT-PCR analysis of IFNGR1/IFNGR2 mRNA in CRC cells transfected with GFP or lncRNA 60967.1 vector. ( K ) Western blot analysis of IFN-γRa protein in CRC cells (DLD1, HCT8, and SW480) transfected with GFP or lncRNA 60967.1 vector. ( L ) qRT-PCR evaluation of IFNGR1/IFNGR2 mRNA in CRC cells (DLD1, HCT8, and SW480) stimulated by ATRA or DMSO. Abbreviation: CCK-8, Cell Counting Kit-8. NS: not significant (t-test); ** P < 0.05, *** P < 0.01 (t-test)

Article Snippet: To capture the PLCD4-RNA complex, human PLCD4 antibody (30133-1-AP, Proteintech) and IgG antibody were incubated with Protein A + G beads overnight at 4 °C.

Techniques: Functional Assay, Expressing, Fluorescence, In Situ Hybridization, Quantitative RT-PCR, Immunoprecipitation, Control, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Cell Counting

Journal: Molecular Cancer

Article Title: Integrative multi-omics analysis reveals the LncRNA 60967.1–PLCD4–ATRA axis as a key regulator of colorectal cancer progression and immune response

doi: 10.1186/s12943-025-02359-x

Figure Lengend Snippet: PLCD4 acts as a tumor suppressor in CRC. ( A ) Migration and invasion assays using two CRC cell lines (HCT116 and DLD1) transfected with either GFP (NC) or PLCD4 expression (PLCD4 OE) vectors. ( B ) Tumors from Balb/c nude mice injected with HCT116 cells transduced with either GFP (Top) or PLCD4 (Bottom) expression vectors. ( C ) Tumor growth over time in Balb/c nude mice injected with HCT116 cells transduced with GFP (blue) or PLCD4 (pink) expression vectors. ( D ) Measurement of tumor weight in Balb/c nude mice injected with HCT116 cells transduced with GFP (blue) or PLCD4 (pink) expression vectors. ( E ) Western blot analysis of PLCD4 expression in CRC cell lines (DLD1, SW480, HCT116) following siRNA-mediated knockdown (siPLCD4#1 or siPLCD4#2) or non-targeting control siRNA (siControl). ( F ) Migration and invasion assays using CRC cell lines (DLD1, SW480, HCT116) transfected with two PLCD4-targeting siRNAs (siPLCD4#1 or siPLCD4#2) or non-targeting control siRNA (siControl)

Article Snippet: To capture the PLCD4-RNA complex, human PLCD4 antibody (30133-1-AP, Proteintech) and IgG antibody were incubated with Protein A + G beads overnight at 4 °C.

Techniques: Migration, Transfection, Expressing, Injection, Transduction, Western Blot, Knockdown, Control

Journal: Molecular Cancer

Article Title: Integrative multi-omics analysis reveals the LncRNA 60967.1–PLCD4–ATRA axis as a key regulator of colorectal cancer progression and immune response

doi: 10.1186/s12943-025-02359-x

Figure Lengend Snippet: LncRNA 60967.1 overexpression promotes PLCD4 and ATRA expression, enhances immune cell infiltration, and reduces tumor growth in mouse CRC models. ( A1–A2 ) qRT-PCR analysis of lncRNA 60967.1, PLCD4, and IFNGR1/2 expression in tumor tissues from mice injected with mouse CRC cells (CT26, A1 ; MC38, A2 ) transfected with lncRNA 60967.1 or GFP vectors. (B1–B2) Western blot analysis of PLCD4 and IFN-γRα proteins in tumor tissues from mice injected with CT26 cells ( B1 ) or MC38 cells ( B2 ) transfected with lncRNA 60967.1 or GFP vectors. (C1–C2) ELISA analysis of ATRA in tumor tissues from mice injected with CT26 cells ( C1 ) or MC38 cells ( C2 ) transfected with lncRNA 60967.1 or GFP vectors. (D1–D2) Tumors harvested from Balb/c mice that were subcutaneously injected with either CT26 cells ( D1 ) or MC38 cells ( D2 ). Each cell line was transfected with GFP or lncRNA 60967.1 vectors prior to injection, allowing comparison of tumor growth and size between the control and experimental groups. (E1–E2) Tumor growth curves in Balb/c mice injected with CT26 cells ( E1 ) or MC38 cells ( E2 ) transfected with either GFP or lncRNA 60967.1 vectors. Tumor sizes were measured at regular intervals to compare progression between the control (GFP) and lncRNA 60967.1 overexpression groups. (F1–F2) Measurement of tumor weight in Balb/c nude mice injected with CT26 cells ( F1 ) or MC38 cells ( F2 ), transfected with either GFP or lncRNA 60967.1 expression vectors. ( G ) IHC analysis of CD4 and CD8 expression in tumor tissues from Balb/c mice subcutaneously injected with either CT26 or MC38 cells. Each cell line was transfected with GFP or lncRNA 60967.1 vectors before injection, enabling a comparison of immune cell infiltration between control and lncRNA 60967.1-overexpressing groups. ( H ) IHC analysis of IFN-γ, PD-1, PD-L1, and Ki67 expression in tumor tissues from Balb/c mice subcutaneously injected with either CT26 or MC38 cells. Each cell line was transfected with GFP or lncRNA 60967.1 vectors before injection, allowing for a comparison of the functional states or activation/exhaustion profiles of immune cells between the control and lncRNA 60967.1-overexpressing groups. NS indicates no significant difference (t-test); ** P < 0.05, *** P < 0.01 (t-test)

Article Snippet: To capture the PLCD4-RNA complex, human PLCD4 antibody (30133-1-AP, Proteintech) and IgG antibody were incubated with Protein A + G beads overnight at 4 °C.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Injection, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison, Control, Functional Assay, Activation Assay

Journal: Molecular Cancer

Article Title: Integrative multi-omics analysis reveals the LncRNA 60967.1–PLCD4–ATRA axis as a key regulator of colorectal cancer progression and immune response

doi: 10.1186/s12943-025-02359-x

Figure Lengend Snippet: LncRNA 60967.1 upregulates immune checkpoint gene expression and enhances anti–PD-1 efficacy in CRC mouse models. ( A1-A3 ) qRT-PCR analysis of PD-L1, CD47, and CD276 mRNA levels in three CRC cell lines (DLD1, A1 ; HCT8, A2 ; and SW480, A3 ) transfected with either a GFP control or an lncRNA 60967.1 expression vector. ( B ) Western blot analysis of PD-L1 levels in DLD1, HCT8, and SW480 cells transfected with either a GFP control or a lncRNA 60967.1 expression vector. ( C ) qRT-PCR analysis of lncRNA 60967.1, PLCD4, IFNGR1, and IFNGR2 expression in HCT8 cells transfected with an siRNA vector targeting lncRNA 60967.1 (HCT8-siRNA-1) or a non-targeting control vector (HCT8-NC). ( D ) qRT-PCR validation of PD-L1, CD47, and CD276 mRNA expression in HCT8 cells transfected with siRNA targeting lncRNA 60967.1 (HCT8-siRNA-1) or a non-targeting control (HCT8-NC). ( E ) Western blot analysis of PD-L1 expression in HCT8 cells transfected with siRNA targeting lncRNA 60967.1 (HCT8-siRNA-1) or a non-targeting control (HCT8-NC). (F1–F2) qRT-PCR measurement of immune checkpoint gene mRNA levels (PD-L1, CD47, and CD276) in tumor tissues from Balb/c mice injected with CT26 cells ( F1 ) or MC38 cells ( F2 ). Cell lines were transfected with GFP (control) or lncRNA 60967.1 vectors before injection to compare checkpoint gene expression. ( G ) Tumors harvested from Balb/c mice that were subcutaneously injected with CT26 cells transfected with either GFP (Top) or lncRNA 60967.1 vectors (Bottom), followed by intraperitoneal administration of anti–PD-1 monoclonal antibody. ( H ) Tumor growth over time in Balb/c nude mice injected with CT26 cells transfected with either GFP or lncRNA 60967.1 vectors, followed by intraperitoneal administration of anti-PD-1 monoclonal antibody. ( I ) Tumor weight in Balb/c nude mice injected with CT26 cells transfected with either GFP or lncRNA 60967.1 vectors, followed by intraperitoneal administration of anti-PD-1 monoclonal antibody. NS indicates no significant difference (t-test); ** P < 0.05, *** P < 0.01 (t-test)

Article Snippet: To capture the PLCD4-RNA complex, human PLCD4 antibody (30133-1-AP, Proteintech) and IgG antibody were incubated with Protein A + G beads overnight at 4 °C.

Techniques: Gene Expression, Quantitative RT-PCR, Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Biomarker Discovery, Injection

Journal: Molecular Cancer

Article Title: Integrative multi-omics analysis reveals the LncRNA 60967.1–PLCD4–ATRA axis as a key regulator of colorectal cancer progression and immune response

doi: 10.1186/s12943-025-02359-x

Figure Lengend Snippet: A proposed model suggests that lncRNA 60967.1 plays a regulatory role in modulating the PLCD4/ATRA axis and anti-PD-1 therapy, influencing immune responses and affecting CRC progression

Article Snippet: To capture the PLCD4-RNA complex, human PLCD4 antibody (30133-1-AP, Proteintech) and IgG antibody were incubated with Protein A + G beads overnight at 4 °C.

Techniques:

Journal: Scientific reports

Article Title: Phospholipase C-related catalytically inactive protein regulates cytokinesis by protecting phosphatidylinositol 4,5-bisphosphate from metabolism in the cleavage furrow.

doi: 10.1038/s41598-019-49156-3

Figure Lengend Snippet: Figure 5. PRIP regulates the accumulation of RhoA and coordinates PI(4,5)P2 levels at the cleavage furrow. (a,b) Transfection with PRIP2-si1 or PRIP2-si2 inhibits RhoA accumulation at the cleavage furrow in HeLa cells. Representative images in a stage of cytokinesis (cytokinesis entry and early cytokinesis) are shown in (a). RhoA signals were visualised using a specific antibody. Arrowheads in (a) indicate abnormal localisation of RhoA. Graph in (b) shows the fluorescence intensity (arbitrary units) of RhoA at the cleavage furrow in early cytokinesis. The data are presented as the mean ± SD (n = 45 for each group). **p < 0.01, ***p < 0.001 (Kruskal–Wallis test followed by Dunn’s multiple comparison test). (c–f) PRIP2 silencing inhibits PI(4,5)P2 accumulation at the cleavage furrow in HEK293 cells. HEK293 cells stably expressing EGFP-tagged pleckstrin homology domain of PLCδ1 (EGFP-PLCδPH) were transfected with the indicated siRNAs (e,f) together with Halo-tagged vector (Halo-empty) or Halo-tagged PRIP1 (Halo-PRIP1) (e,f). Analyses of EGFP intensity of the cells were performed by flow cytometry (c). The cells were trypsinised and observed (d). (e,f) Representative images of EGFP-PLCδPH and Halo-tagged proteins in a stage of cytokinesis are shown in (e). Graph in (f) presents the mean intensity of EGFP in the cleavage furrow ± SD (n > 60 for each group). ***p < 0.001 (Kruskal–Wallis test followed by Dunn’s multiple comparison test).

Article Snippet: Anti-RhoA (sc-418) and anti-PLCδ1 (D-7) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transfection, Fluorescence, Comparison, Stable Transfection, Expressing, Plasmid Preparation, Flow Cytometry

Journal: Circulation Research

Article Title: Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

doi: 10.1161/CIRCRESAHA.120.317295

Figure Lengend Snippet: Protein interaction network analysis reveals numerous interactions of proteins that are differentially abundant in aortas of SR-uPA +/0 mice. A protein-protein relational network was built based on experimentally validated direct interactions. The network is comprised of 87 proteins, each portrayed as a circular node (all nodes are identified in Data Set V in the Data Supplement ). Key highly connected nodes (hubs) are labeled together with 2 members of the matrix metalloproteinase family of extracellular proteases and several extracellular matrix components. ACTB indicates beta actin; AGRN, agrin; BCAM, basal cell adhesion molecule; ELN, elastin; FBLN5, fibulin 5; FN1, fibronectin 1; HSPG2, heparan sulfate proteoglycan 2; LAMA5, laminin subunit alpha 5; LAMB2, laminin subunit beta 2; LAMC1, laminin subunit gamma 1; LTBP4, latent transforming growth factor binding protein 4; MMP2, matrix metalloproteinase 2; MMP3, matrix metalloproteinase 3; MYH9, myosin heavy chain 9; NID1, nidogen1; NID2, nidogen 2; and PLAU, urokinase-type plasminogen activator.

Article Snippet: HSPG2 was visualized with 0.375 μg/mL of HSPG2 antibody (PB9277; Boster Biological Technology) in 5% milk/PBST (phosphate-buffered saline/Tween) overnight.

Techniques: Labeling, Binding Assay