Journal: Traffic (Copenhagen, Denmark)

Article Title: PLCγ1-PKCγ signaling-mediated Hsp90α plasma membrane translocation facilitates tumor metastasis.

doi: 10.1111/tra.12179

Figure Lengend Snippet: Figure 3: PLC𝛄1 regulation in plasma membrane translocation of Hsp90𝛂. A) Signaling pathways regulating membrane translocation of Hsp90α were screened using inhibitors of PLC, PI3K, Src and Erk. Cell-surface expression of Hsp90α was detected by confocal imaging. Actin filaments (red), Hsp90α on the cell surface (green) and DAPI (blue). Scale bar: 50 μm. B) Plasma membrane extractions of tumor cells pretreated with the inhibitors of PLC, PI3K, Src and Erk were separated with 12% SDS–PAGE and blotted with anti-Hsp90α antibody. C) Activation of PLCγ1 was detected after treating the cells with the PLC inhibitor U73122 (PLCi). D) Immunofluorescence analysis of cell-surface Hsp90α by confocal imaging, on cells treated with PLCγ1 siRNA. Actin filaments (red), Hsp90α on the cell surface (green) and DAPI (blue). Scale bar: 100 μm. E) Membrane extraction of tumor cells pretreated with PLCγ1 siRNA was detected by western blotting with anti-Hsp90α antibody. F) Cells were pretreated with PLCγ1 siRNA. Lysates were immunoprecipitated with anti-Hsp90α antibody. Western blotting analyses of IP samples were performed using a phospho-Thr mouse mAb. G) Levels of plasma membrane Hsp90α and total PLCγ1 in lysates of MDA-MB-231 and SKBr-3 cells were detected by western blotting.

Article Snippet: PKCγ siRNA was purchased from Santa Cruz.

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Protein-Protein interactions, Expressing, Imaging, SDS Page, Activation Assay, Extraction, Western Blot, Immunoprecipitation

Journal: Traffic (Copenhagen, Denmark)

Article Title: PLCγ1-PKCγ signaling-mediated Hsp90α plasma membrane translocation facilitates tumor metastasis.

doi: 10.1111/tra.12179

Figure Lengend Snippet: Figure 4: PKC𝛄mediation of PLC𝛄1’s function in regulating cell plasma membrane translocation of Hsp90𝛂. A) Western blotting analysis of plasma membrane Hsp90α after cells were treated with different doses of the intracellular Ca2+ chelator BAPTA-AM. B) Membrane Hsp90α was detected by western blotting analysis of cells treated with the PKC inhibitor chelerythrine chloride (100 nM, 1 μM) or activator PMA (100 nM, 1 μM). C) Cells were pretreated with PLC inhibitor or PKC inhibitor, and then EGF was added. Lysates were immunoprecipitated with anti-Hsp90α antibody. Western blotting analyses of IP samples were performed using a phospho-Thr mouse mAb. D) Representative images of membrane translocation of Hsp90α in MDA-MB-231 cells. Cell-surface Hsp90α was detected by confocal imaging. Actin filaments (red), Hsp90α on the cell surface (green) and DAPI (blue). Scale bar: 100 μm. E) Analysis of membrane-localized Hsp90α in cells transduced with PKCγ siRNA or PKCγ plasmid; 48 h after treatment, cell lysates and plasma membrane extracts were analyzed by western blotting. Scramble RNA or vector was used as negative controls for siRNA or plasmid experiments, respectively. F) Cells transduced with PKCγ siRNA or PKCγ plasmid; 48 h after treatment, cell lysates were detected. G) Analysis of membrane Hsp90α after treatment with Rab11 siRNA (Rab11A and Rab11B). Scramble RNA was used as the control. H) Levels of Hsp90α in plasma membranes were detected by western blotting after cells were treated with Rab11 siRNA and PKCγ plasmid.

Article Snippet: PKCγ siRNA was purchased from Santa Cruz.

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Western Blot, Immunoprecipitation, Imaging, Transduction, Plasmid Preparation, Control

Journal: Toxics

Article Title: Protein Modifications and Metabolic Alterations in the Rat Striatum Following Oil Mist Particulate Matter Exposure Revealed via Untargeted Metabolomics and Phosphoproteomics

doi: 10.3390/toxics14030249

Figure Lengend Snippet: Validation and analysis of key phosphorylated protein levels: ( A ) Representative WB images of total and phosphorylated forms of CALM3, CaMK2b, GSK-3β, PRKCG, and TH in rat striatal tissues. ( B ) Quantitative analysis of band intensities shown in ( A ); relative phosphorylation level = (phosphorylated protein/GAPDH)/(total protein/GAPDH). * p < 0.05 and ** p < 0.01.

Article Snippet: The membranes were blocked with nonfat milk and incubated overnight at 4 °C with primary antibodies against p-CALM3 (1:500, Affinity, Cincinnati, OH, USA), p-CaMK2b (1:500, Affinity, Cincinnati, OH, USA), p-GSK-3β (1:1000, Proteintech, Rosemont, IL, USA), p-PRKCG (1:500, Affinity, Cincinnati, OH, USA), p-TH (1:500, Affinity, Cincinnati, OH, USA), CALM3 (1:1000, Proteintech, Rosemont, IL, USA), CaMK2b (1:1000, Proteintech, Rosemont, IL, USA), GSK-3β (1:1000, Proteintech, Rosemont, IL, USA), PRKCG (1:1000, Proteintech, Rosemont, IL, USA), and TH (1:1000, Proteintech, Rosemont, IL, USA).

Techniques: Biomarker Discovery, Phospho-proteomics

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Diacylglycerol kinase γ facilitates the proliferation and migration of neural stem cells in the developing neural tube

doi: 10.3724/abbs.2024156

Figure Lengend Snippet: Immunofluorescence staining showing the coexpression of DGKγ with p-PKCγ or p-PKCδ DGKγ was coexpressed with p-PKCγ (A) and p-PKCδ (B) in the cytoplasm and nucleus of neuroepithelial NT cells at E11.5. Blue: DAPI; red: DGKγ; green: p-PKCγ or p-PKCδ. Scale bar: 100 μm. n = 5.

Article Snippet: The PVDF membranes were subsequently incubated with primary antibodies against DGKγ (1:500, ab89037; Abcam, Cambridge, UK), p-PKCδ (1:1000, sc-365969; Santa Cruz Biotech), PKCδ (1:1000, sc-213; Santa Cruz Biotech), p-PKCγ (1:1000, 44-975G; Invitrogen), PKCγ (1:1000, BM5623; Boster), and β-actin (1:4000, AP0060; Bioworld Technology, Shanghai, China) overnight at 4°C.

Techniques: Immunofluorescence, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Diacylglycerol kinase γ facilitates the proliferation and migration of neural stem cells in the developing neural tube

doi: 10.3724/abbs.2024156

Figure Lengend Snippet: Changes in DAG content and p-PKC subtypes during NSC migration (A) DAG contents were determined by ELISA, which revealed that the DAG contents were significantly increased in sh-DGKγ-treated cells compared with those in the sh-NC-treated cells and significantly increased in R59949- and PMA-treated cells compared with those in the vehicle-treated cells. (B) Representative western blots of p-PKCγ and p-PKCδ in NSCs were quantified via western blotting. (C,D) Densitometric analysis of the western blots revealed that the p-PKCγ/PKCγ ratio was significantly lower in the PMA group than in the vehicle group; however, the p-PKCδ/PKCδ ratio was significantly greater in the sh-DGKγ group than in the sh-NC group, which was also significantly greater in the R59949 and PMA groups than in the vehicle group. *P < 0.05, **P < 0.01, ***P < 0.001 vs sh-NC or vehicle. n = 3.

Article Snippet: The PVDF membranes were subsequently incubated with primary antibodies against DGKγ (1:500, ab89037; Abcam, Cambridge, UK), p-PKCδ (1:1000, sc-365969; Santa Cruz Biotech), PKCδ (1:1000, sc-213; Santa Cruz Biotech), p-PKCγ (1:1000, 44-975G; Invitrogen), PKCγ (1:1000, BM5623; Boster), and β-actin (1:4000, AP0060; Bioworld Technology, Shanghai, China) overnight at 4°C.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Diacylglycerol kinase γ facilitates the proliferation and migration of neural stem cells in the developing neural tube

doi: 10.3724/abbs.2024156

Figure Lengend Snippet: Schematic diagram of the role of DGKγ in NSC migration and proliferation A schematic diagram showing that the DAG/PKCδ signaling pathway is involved in NSC migration and that the mTOR signaling pathway, independent of PKC, may be involved in NSC proliferation.

Article Snippet: The PVDF membranes were subsequently incubated with primary antibodies against DGKγ (1:500, ab89037; Abcam, Cambridge, UK), p-PKCδ (1:1000, sc-365969; Santa Cruz Biotech), PKCδ (1:1000, sc-213; Santa Cruz Biotech), p-PKCγ (1:1000, 44-975G; Invitrogen), PKCγ (1:1000, BM5623; Boster), and β-actin (1:4000, AP0060; Bioworld Technology, Shanghai, China) overnight at 4°C.

Techniques: Migration