beta pan pkc  (Proteintech)

Journal: Antioxidants (Basel, Switzerland)

Article Title: Prevention of LPS-Induced Acute Kidney Injury in Mice by Bavachin and Its Potential Mechanisms.

doi: 10.3390/antiox11112096

Figure Lengend Snippet: Figure 4. Bavachin decreases PKCβ activation and NOX4 expression in LPS-induced AKI mice and LPS-treated HK-2 cells. (A) Immunohistochemical detection of phospho-protein kinase C β (P-PKCβ) and NADPH oxidase 4 (NOX4) in kidney tissues using DAB. Nuclei were counterstained with hematoxylin (original magnification, 200×; scale bars, 20 µm; n = 3 mice per group). (B) HK-2 cells were pretreated with bavachin 0.1 µg/mL for 1 h and then treated with 1 µg/mL LPS for 1 h. The protein levels of p-PKCβ and NOX4 were analyzed by Western blotting, quantified using ImageJ software, and normalized to PCKβ and β-actin, respectively (n = 3). Data are presented as mean ± SEM. ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. LPS.

Article Snippet: The following primary antibodies were used: NGAL (ab63929; Abcam, Cambridge, UK), KIM-1 (NBP1-76701; Novus Biologicals, Centennial, CO, USA), NADPH oxidase 4 (NOX4, ab109225; Abcam), malondialdehyde (MDA, ab243066; Abcam), Krueppel-like factor 5 (KLF5, ab137676; Abcam), 4-hydroxynonenal (4-HNE, MAB3249; R & D Systems, Minneapolis, MN, USA), protein kinase C (PKC) β (#46809; Cell Signaling Technology, Inc., Danvers, MA, USA), and phospho-PKC (p-PKC) β (#9371; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Expressing, Immunohistochemical staining, Western Blot, Software, Control

Journal: Molecular cell

Article Title: Protein Kinase C Quality Control by Phosphatase PHLPP1 Unveils Loss-of-Function Mechanism in Cancer

doi: 10.1016/j.molcel.2019.02.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PKCα/β pSer 657/660 , Cell Signaling Technologies , 9371S.

Techniques: Transduction, Western Blot, Immunoprecipitation, Saline, Modification, Labeling, BIA-KA, Autoradiography, Recombinant, Plasmid Preparation, Software

Journal: Antioxidants & Redox Signaling

Article Title: Isosteviol Protects Free Fatty Acid- and High Fat Diet-Induced Hepatic Injury via Modulating PKC-β/p66Shc/ROS and Endoplasmic Reticulum Stress Pathways

doi: 10.1089/ars.2018.7521

Figure Lengend Snippet: The role of PKC-β activation in PA-induced hepatic injury. (A) Rat primary hepatocytes were treated with PA (1 mM) with or without ISV (3 or 10 μM) for 24 h. The protein levels of cytosolic phosphorylated PKC-β (p-PKC-β) and total PKC-β (t-PKC-β) were determined by Western blot analysis. Representative immunoblot images are shown. β-Actin was used as loading control. Relative protein levels of p-PKC-β and t-PKC-β were calculated based on the mean ± SD of three independent experiments. Statistical significance compared with control or PA group, *p < 0.05, **p < 0.01. (B) The effect of ISV and RBX on PKC-β enzyme activity was measured by using an in vitro luciferase assay as described in the Materials and Methods section. The IC50 of ISV and RBX were calculated by using Sigma plot software. (C–F) Rat primary hepatocytes were treated with PA (1 mM) with vehicle control, ISV (10 μM), or RBX (20 nM) for 24 h. (C) The apoptotic cells were measured by flow cytometry as described in the Materials and Methods section. (D) The intracellular lipid accumulation was measured by Nile red staining and flow cytometry. Annexin V positive cells ratio and Nile red fluorescence intensity were calculated, respectively, based on the mean ± SEM of three independent experiments. (E, F) Representative immunoblot images of phosphorylated p66Shc (p-p66Shc) and total p66Shc (t-p66Shc) in total cell lysate, cytosol, and mitochondria are shown. β-Actin and Cox4i1 were used as loading control for cytosolic and mitochondrial protein, respectively. Relative protein levels of p-p66Shc and p66Shc are presented based on the mean ± SD of three independent experiments. Statistical significance compared with control or PA group, *p < 0.05, **p < 0.01, ***p < 0.001, and NS, no significance. PKC-β, protein kinase C-β; RBX, Ruboxistaurin. Color images are available online.

Article Snippet: Antibodies against phospho-PKC-β (9371), Pin1 (3722), XBP1s (12782), IP3R1 (8568), JNK (9252), phospho-JNK (9255), p38 (9212), phospho-P38 (9216), PERK (3192), phospho-PERK (3179), phospho-eIF2α (9721), eIF2α (2103), anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7076), and anti-rabbit IgG, HRP-linked antibody (7074) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Western Blot, Control, Activity Assay, In Vitro, Luciferase, Software, Flow Cytometry, Staining, Fluorescence

Journal: Antioxidants & Redox Signaling

Article Title: Isosteviol Protects Free Fatty Acid- and High Fat Diet-Induced Hepatic Injury via Modulating PKC-β/p66Shc/ROS and Endoplasmic Reticulum Stress Pathways

doi: 10.1089/ars.2018.7521

Figure Lengend Snippet: Effect of PKC-β on PA-induced hepatic injury in rat primary hepatocytes. Rat primary hepatocytes were transduced with lentiviral PKC-β shRNAs for 48 h as described in the Materials and Methods section. (A) Representative immunoblots of PKC-β and β-actin are shown. Relative protein levels of PKC-β were determined based on the mean ± SD of three independent experiments, and β-actin was used as an internal loading control. Statistical significance compared with control group, **p < 0.01, ***p < 0.001. (B–D) Rat primary hepatocytes were further treated with PA (1 mM) with or without ISV (10 μM) or RBX (20 nM) for 24 h. (B) The intracellular lipid, (C) cell apoptosis, ROS levels, and MTP were measured by flow cytometry as previously described. Nile red fluorescence intensity, Annexin V positive cells ratio, high DCF intensity cells ratio, and JC-1 aggregates were calculated, respectively, based on the mean ± SEM of three independent experiments. Statistical significance compared with control-shRNA or PA group, *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Representative immunoblot images of p66Shc in cytosol and mitochondria are shown. Cox4i1 and β-actin were used as internal loading controls for mitochondria and cytosol, respectively. Relative protein levels of p66Shc represent the mean ± SD of three independent experiments. Statistical significance compared with control-shRNA or PA group, *p < 0.05, **p < 0.01. (E) Rat primary hepatocytes were treated with 50 nM dPPA with or without PA and ISV (0, 1, 3, and 10 μM) for 24 h. Hepatocyte apoptosis was measured by flow cytometry. Annexin V positive cells ratio was calculated based on the mean ± SEM of three independent experiments. Statistical significance compared with PA group, *p < 0.05, **p < 0.01, ***p < 0.001, and NS, no significance. dPPA, 12-deoxyphorbol 13-phenylacate 20-acetate. Color images are available online.

Article Snippet: Antibodies against phospho-PKC-β (9371), Pin1 (3722), XBP1s (12782), IP3R1 (8568), JNK (9252), phospho-JNK (9255), p38 (9212), phospho-P38 (9216), PERK (3192), phospho-PERK (3179), phospho-eIF2α (9721), eIF2α (2103), anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7076), and anti-rabbit IgG, HRP-linked antibody (7074) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Transduction, Western Blot, Control, Flow Cytometry, Fluorescence, shRNA

Journal: Antioxidants & Redox Signaling

Article Title: Isosteviol Protects Free Fatty Acid- and High Fat Diet-Induced Hepatic Injury via Modulating PKC-β/p66Shc/ROS and Endoplasmic Reticulum Stress Pathways

doi: 10.1089/ars.2018.7521

Figure Lengend Snippet: Effect of ISV and RBX on HFD-induced liver injury in rats. Rats were fed with regular rodent diet or HFD for 4 weeks and then, the HFD-fed group rats were treated with ISV (20 mg/kg) or RBX (40 μg/kg) by oral gavage daily for 4 weeks. (A) Representative images of H&E staining of the liver tissues from each treatment group are shown. N = 6–8 rats per group. (B) The serum lipid levels. (C) The enzyme activities of ALT and AST in the serum. (D) Hepatic SOD activity normalized with total protein amount. (E) Hepatic MDA levels normalized with total protein amount. The values are presented as mean ± SD. N = 6–8 rats per group. *p < 0.05, **p < 0.01, ***p < 0.001, and NS, no significance. (F) Representative immunoblot images of p-PKC-β, PKC-β, p-p66Shc, p66Shc, and β-actin are shown. (G) Representative immunoblot images of PACS-2, IP3R1, and β-actin are shown. (H) Representative images of electron microscopy showing mitochondria-associated ER membrane. The white arrows present the increased ER-mitochondria interaction. (I) Representative immune blot images of cytosolic stress-responsive genes and nuclear ER stress genes, including p-JNK, JNK, p-p38, p38, p-PERK, PERK, p-eIF2α, GRP78, ATF4, and CHOP. Actin and Lamin B were used as loading controls for cytosolic and nuclear proteins, respectively. IP3R1, inositol 1,4,5-trisphosphate receptor type 1; JNK, c-Jun N-terminal kinase; PACS-2, phosphofurin acidic cluster sorting protein 2. Color images are available online.

Article Snippet: Antibodies against phospho-PKC-β (9371), Pin1 (3722), XBP1s (12782), IP3R1 (8568), JNK (9252), phospho-JNK (9255), p38 (9212), phospho-P38 (9216), PERK (3192), phospho-PERK (3179), phospho-eIF2α (9721), eIF2α (2103), anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7076), and anti-rabbit IgG, HRP-linked antibody (7074) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Staining, Activity Assay, Western Blot, Electron Microscopy, Membrane

Journal: Antioxidants & Redox Signaling

Article Title: Isosteviol Protects Free Fatty Acid- and High Fat Diet-Induced Hepatic Injury via Modulating PKC-β/p66Shc/ROS and Endoplasmic Reticulum Stress Pathways

doi: 10.1089/ars.2018.7521

Figure Lengend Snippet: Schematic diagram of the potential signaling pathways involved in ISV-mediated protective effect on HFD-/FFAs-induced hepatic lipotoxic injury. HFD feeding increases FFAs in circulation, which upregulates p66Shc expression and activation of PKC-β. Activated PKC-β phosphorylates p66Shc, allowing its recognition by Pin1 and dephosphorylation by PP2A and the transfer from the cytosol to the mitochondrion, where p66Shc induces free O2 production and ROS and results in cytochrome c release. ROS can further activate PKC-β. In the meantime, FFAs also induces ER calcium depletion by upregulating IP3R1/PACS-2 and activates the UPR. Increased cytosolic cytochrome c and ROS and upregulation of ATF4/CHOP promote cell apoptosis. ISV not only inhibits ROS and ER stress but also inhibits PKC-β activation, which prevents mitochondrial translocation of p66Shc and inhibits ROS production. In addition, ISV also inhibits p66Shc expression. Cyto-c, cytochrome c; Pin1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1; PP2A, serine/threonine protein phosphatase 2A; TOM, translocase of the outer membrane; UPR, unfolded protein response. Color images are available online.

Article Snippet: Antibodies against phospho-PKC-β (9371), Pin1 (3722), XBP1s (12782), IP3R1 (8568), JNK (9252), phospho-JNK (9255), p38 (9212), phospho-P38 (9216), PERK (3192), phospho-PERK (3179), phospho-eIF2α (9721), eIF2α (2103), anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7076), and anti-rabbit IgG, HRP-linked antibody (7074) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, De-Phosphorylation Assay, Translocation Assay, Membrane