Journal: Experimental and therapeutic medicine

Article Title: Enhancement of ICAM-1 via the JAK2/STAT3 signaling pathway in a rat model of severe acute pancreatitis-associated lung injury.

doi: 10.3892/etm.2016.2988

Figure Lengend Snippet: Figure 7. Representative immunohistochemical results for JAK2 in the lung tissue sections from the (A) CON, (B) SAP, and (C) DEX‑treated groups at 24 h following induction of SAP (magnification, x40). JAK2, janus kinase 2; SAP, severe acute pancreatitis; CON, control; DEX, dexamethasone.

Article Snippet: Subsequently, the lung tissue sections were incubated for 1 h with rabbit anti-rat JAK2 (1:50; sc-294) and STAT3 (1:50; sc-482) antibodies (Santa Cruz Biotechnology, Inc.) at room temperature.

Techniques: Immunohistochemical staining, Control

Journal: The Journal of Biological Chemistry

Article Title: The store-operated Ca 2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2

doi: 10.1016/j.jbc.2023.102882

Figure Lengend Snippet: Cartoon summarizing Orai1α-dependent, agonist-stimulated, NF-κB transcriptional activity. G protein–coupled receptor occupation leads to the activation of phospholipase C (PLC) that generates inositol 1,4,5-trisphosphate (IP 3 ), which, in turn, induces Ca 2+ release from the endoplasmic reticulum (ER). Ca 2+ store depletion leads to Ca 2+ influx via Orai1α, which enhances the Ca 2+ concentration nearby. Orai1α and the Ca 2+ -regulated conventional PKC isoform, PKCβ2, are maintained in close apposition by the scaffold protein AKAP79. Therefore, the rise in cytosolic Ca 2+ concentration nearby the channel results in activation of PKCβ2, which has been reported to lead to the activation of NF-κB via the classical IκB kinase (IKK)–IκB signaling pathway, thus, leading to IκB serine phosphorylation and proteasomal degradation, which releases active, dimeric, NF-κB that translocates to the nucleus and initiates transcription of NF-κB-responsive genes.

Article Snippet: PKCβ shRNA (catalog number: sc-29450-SH; from Santa Cruz Biotechnology).

Techniques: Activity Assay, Activation Assay, Concentration Assay, Phospho-proteomics

Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: (A) GNAQQ209L transfected alone or combined with PKC δ or ε into 293FT cells increased Ras-GTP levels. 293FT cells were transfected with indicated cDNA plasmids for 24 hr. Ras-GTP pull-down was performed and detected by western blot with a pan-Ras antibody. TPA stimulation was used a positive control. Expression levels of GNAQQ209L were monitored with Glu-Glu (EE) tag. PKC δ and ε were detected via HA tags.

Article Snippet: cDNA: HA-PKC βII (catalytic domain) , addgene , 16384.

Techniques: Transfection, Western Blot, Positive Control, Expressing

Journal: Cancer cell

Article Title: RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

doi: 10.1016/j.ccell.2017.04.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: cDNA: HA-PKC βII (catalytic domain) , addgene , 16384.

Techniques: Recombinant, Activation Assay, Microarray, Fractionation, Expressing, Cell Culture

Journal: Pigment Cell & Melanoma Research

Article Title: Functional alterations in protein kinase C beta II expression in melanoma

doi: 10.1111/j.1755-148x.2009.00664.x

Figure Lengend Snippet: Figure 2. Expression of PKC isoform mRNAs in MCs and melanoma cell lines. (A) Total RNA was prepared from three independent normal human MC preparations (1, 2, 3), the HPV E6 ⁄ E7 immortalized PIG1 MC cell line, and five melanoma cell lines. The mRNA level of each PKC isoforms was determined by RT-PCR. GAPDH mRNA was used as a control for RNA integrity. (B) Total RNA prepared from cells in panel A was analyzed by RT-PCR for PKCbI and PKCbII using splice form specific primers.

Article Snippet: The PKCb retrovirus was produced by cloning the full-length human PKCbII cDNA (NM_002738.5, OriGene, Rockville, MD, USA) into the retroviral vector LZRS-Linker in two steps.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Pigment Cell & Melanoma Research

Article Title: Functional alterations in protein kinase C beta II expression in melanoma

doi: 10.1111/j.1755-148x.2009.00664.x

Figure Lengend Snippet: Figure 3. Suppression of melanoma growth in soft agar by PKCb re-expression. (A) WM35 melanoma cells were transduced with control Linker or PKCbII retrovirus, and expression of PKCbII assessed by immunoblotting. (B) WM35 cells transduced with Linker or PKCbII retrovirus were suspended in soft agar and colony formation assessed after 2 weeks. Shown is a photograph of the soft agar colonies. (C) Quantification of the WM35 cell soft agar colonies. Note the significant (P < 0.05) reduction in colony formation by PKCbII.

Article Snippet: The PKCb retrovirus was produced by cloning the full-length human PKCbII cDNA (NM_002738.5, OriGene, Rockville, MD, USA) into the retroviral vector LZRS-Linker in two steps.

Techniques: Expressing, Transduction, Control, Western Blot

Journal: Pigment Cell & Melanoma Research

Article Title: Functional alterations in protein kinase C beta II expression in melanoma

doi: 10.1111/j.1755-148x.2009.00664.x

Figure Lengend Snippet: Figure 5. Mitochondrial localization of PKCbII. (A) 888 melanoma cells were stained with MitoTracker Red and antibodies to either PKCbI or PKCbII, shown in green. Note the co-localization of PKCbII with MitoTracker in the merged conventional fluorescent microscope image. (B) Normal human MCs were stained with MitoTracker Red and antibodies to either PKCbI or PKCbI, shown in green. Note the co-localization of PKCbII with MitoTracker in the merged confocal microscope image.

Article Snippet: The PKCb retrovirus was produced by cloning the full-length human PKCbII cDNA (NM_002738.5, OriGene, Rockville, MD, USA) into the retroviral vector LZRS-Linker in two steps.

Techniques: Staining, Microscopy

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Inhibition of protein kinase C beta phosphorylation activates nuclear factor-kappa B and improves postischemic recovery in type 1 diabetes.

doi: 10.1177/1535370220920832

Figure Lengend Snippet: Figure 6. A PKCb inhibitor Rbx decreased phosphorylation of Ser661 residue of PKCb and increased IjBa degradation. HUVEC were grown in normal, high D-glucose, or high D-glucose þ Rbx with ischemia for 24 h. (a) Western blots of HUVEC grown in different glucose concentrations with ischemia. (b) Autophosphorylated PKCbpSer661 band showed greater intensity in cell lysates with high D-glucose that was decreased in the cells grown in the high D-glucose þ Rbx (20 nM). (c) The IjBa levels (IjBa/b-actin) in the corresponding samples were increased in high D-glucose but were significantly reduced in the cells treated with Rbx. Asterisks denote P < 0.05, and n.s. denotes not signifi- cant; n¼ 4 to 6 per group. PKCb: protein kinase C beta; Rbx: ruboxistaurin.

Article Snippet: The PKCb inhibitor, ruboxistaurin (Rbx; LY333531, Cat# HY-10195B, MedChemExpress, Princeton, NJ, USA), was administered tomice at 1mg/kg/day by oral gavage starting 5 days prior to induction of hind limb ischemia (HLI) and continued through the study.16

Techniques: Phospho-proteomics, Residue, Western Blot

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Inhibition of protein kinase C beta phosphorylation activates nuclear factor-kappa B and improves postischemic recovery in type 1 diabetes.

doi: 10.1177/1535370220920832

Figure Lengend Snippet: Figure 7. PKCb inhibition restores NF-jB activation in hyperglycemic diabetic Akita mice after ischemia. (a) Following an HLI (three days postsurgery), Western blots of the gastrocnemius muscle homogenates from the contralateral non-ischemic hind limb were performed. (b) Quantification of the protein bands showed increased basal levels of PKCbpSer661 in untreated Akita mice compared to the WT C57BL/6J mice. Homogenates from Rbx-treated Akita mice showed significantly decreased PKCbpSer661 levels. (c) Western blots of ischemic limb of untreated and Rbx-treated Akita mice. (d) The protein levels of phosphorylated PKCb (PKCbpSer661) were similar to those of non-ischemic limb samples but significantly decreased in the ischemic limb of Rbx-treated Akita mice. (e) Activation of the NF-jB canonical pathway was measured by Western blots of IjBa protein in ischemic hind limbs of untreated Akita and Rbx-treated Akita mice. (f) The IjBa levels were significantly greater in samples from Akita mice without treatments. In Rbx-treated ischemic limbs of Akita mice, the IjBa levels significantly dropped similar to the PKCbpSer661. Asterisks denote P < 0.05, and n.s. denotes not significant; n¼ 4 to 6 per group. (g) Recovery of blood perfusion after induced HLI in WT (C57BL/6J), vehicle-treated Akita (v- Akita), and Rbx-treated Akita (Rbx-Akita) mice viewed by laser speckle imaging immediately after surgery (day 0), day 3, 1 week, and 2 weeks after surgery. (h) Blood flow in the hind limb was measured in each mouse at multiple time points after induction of ischemia. The extent of perfusion in the operated limb is compared to that of the perfusion in the unoperated limb in the same mouse and expressed as a percentage as shown. Zero on X-axis indicates perfusion immediately after surgery, and n ¼ 5 to 8 mice in each group; asterisks denote P < 0.05. (A color version of this figure is available in the online journal.) PKCb: protein kinase C beta; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Rbx: ruboxistaurin; HLI: hind limb ischemia.

Article Snippet: The PKCb inhibitor, ruboxistaurin (Rbx; LY333531, Cat# HY-10195B, MedChemExpress, Princeton, NJ, USA), was administered tomice at 1mg/kg/day by oral gavage starting 5 days prior to induction of hind limb ischemia (HLI) and continued through the study.16

Techniques: Inhibition, Activation Assay, Western Blot, Imaging

Journal: Experimental biology and medicine (Maywood, N.J.)

Article Title: Inhibition of protein kinase C beta phosphorylation activates nuclear factor-kappa B and improves postischemic recovery in type 1 diabetes.

doi: 10.1177/1535370220920832

Figure Lengend Snippet: Figure 8. Interaction between ischemia and hyperglycemia-induced signaling in NF-jB activation and perfusion recovery in PAD. Both ischemia and hyperglycemia induce signaling to activate NF-jB that is required for perfusion recovery. However, chronic hyperglycemic conditions attenuate the ischemia-induced activation of the canonical NF-jB pathway and contribute to poor perfusion recovery, through the effects of increased phosphorylation of PKCb (PKCb-pSer661). Inhibition of PKCb activity by the specific inhibitor ruboxistaurin (Rbx) restores the canonical NF-jB pathway and improves perfusion recovery in diabetic mice after PAD. (A color version of this figure is available in the online journal.) PKCb: protein kinase C beta; NF-jB: nuclear factor-kappa B.

Article Snippet: The PKCb inhibitor, ruboxistaurin (Rbx; LY333531, Cat# HY-10195B, MedChemExpress, Princeton, NJ, USA), was administered tomice at 1mg/kg/day by oral gavage starting 5 days prior to induction of hind limb ischemia (HLI) and continued through the study.16

Techniques: Activation Assay, Phospho-proteomics, Inhibition, Activity Assay