Journal: Frontiers in Oncology

Article Title: Suppression of EGFR/PKC-δ/NF-κB Signaling Associated With Imipramine-Inhibited Progression of Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2021.735183

Figure Lengend Snippet: The inhibition of EGFR/PKC-δ/NF-κB proteins phosphorylation and induction of apoptosis-related proteins by imipramine in CL1-5-F4/ NF-κB-luc2 bearing tumor. (A–C) The protein expression from IHC of EGFR (Try 1068), PKC-δ (Thr507), NF-κB (Ser536), cleaved caspase-3, -8, -9, MMP-9, XIAP, MCL-1 and their quantification bar chart are presented. (D, E) The tumor ex vivo Western blotting from each mice of cleaved caspase-3, -8, -9 and PARP-1 is presented. ( ** p < 0.01 vs . vehicle; scale bar =100 μm).

Article Snippet: Primary antibodies against Matrix metalloproteinase-9 (MMP-9) (AB19016, Millipore), vascular endothelial growth factor (VEGF) (ab1316, Abcam, Cambridge, UK), EGFR (Try 1068) (#2234, Cell signaling, Danvers, MA, USA), EGFR (E-AB-63555, Elabscience, Houston, TX, USA), PKC-δ (Thr507) (E-AB-20968, Elabscience), PKC-δ (E-AB-14675, Elabscience), NF-κB p65 (Ser536) (E-AB-70335, Elabscience), NF-κB p65 (E-AB-22066, Elabscience), cell leukemia-1 (MCL-1) (BV-438, BioVision), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) (D16A8, Cell signaling), X-linked inhibitor of apoptosis protein (XIAP) (PA5-29253, Thermo Fisher Scientific), Fas (E-AB-40063, Elabscience), Fas ligand (FasL) (E-AB-31410, Elabscience), cleaved caspase-3 (E-AB-30004, Elabscience), cleaved caspase-8 (E-AB-22107, Elabscience), cleaved caspase-9 (#9505, Cell Signaling Technology), PARP-1 (#9532, Cell Signaling Technology) and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, Waltam, MA, USA) for Western blotting were purchased from different companies as listed.

Techniques: Inhibition, Phospho-proteomics, Expressing, Ex Vivo, Western Blot

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 δ(kinase dead) , addgene , 16389.

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 δ(kinase dead) , addgene , 16389.

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

Journal: The Journal of veterinary medical science

Article Title: An immunohistochemical study of protein kinase C in the bovine retina.

doi: 10.1292/jvms.68.71

Figure Lengend Snippet: Fig. 1. Western blot analysis of PKC isoforms from three samples of bovine retinas. A single immunoreactive band of PKC α (82 kDa) was detected. PKC βI antisera recognized two bands, including a major band at 80 kDa and a minor band between 51 and 36 kDa. Intense immunoreactive bands (arrowhead) of PKC δ and PKC θ were detected between 51 kDa and 36 kDa, respectively. A tiny band at the expected size (arrow) that reacted with PKC δ and θ antisera was found in the same lane. A lowest line in photo represents signals of β-actin in each lane. Molecular sizes were determined from stan- dard proteins (194 kDa, 116 kDa, 51 kDa, 36 kDa, and 28 kDa, in order).

Article Snippet: Rabbit polyclonal antisera to PKC α, PKC βI, PKC δ, and PKC θ were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pharmacological inhibitors of PKC β and PKC δ suppressed CSE-induced p66Shc activation and its mitochondrial translocation in airway epithelial cells. The total and phosphorylated protein expression of PKC β and PKC δ was notably increased in Beas-2b cells exposed to 7.5% CSE for 24 h, and these alterations could be downregulated by 30 min pretreatments with 10 μ M LY333531 and 5 μ M rottlerin. A representative band is shown for each condition (a), and densitometric analysis of the bands was carried out by Bandscan 5.0 software to quantify the protein expression (b). Further, Western blot analysis revealed that the pretreatments with LY333531 and rottlerin significantly decreased the protein expression of p66Shc and p-p66Shc in the whole cell lysates (c) and suppressed p66Shc mitochondrial translocation and cytochrome c release from the mitochondria (d) in Beas-2b cells incubated with 7.5% CSE for 24 h. All the data are shown as the mean ± SD from three independent experiments. ∗ P < 0.05 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: The membranes were then incubated with specific antibodies against p66Shc (1 : 1000, Abcam), p-p66Shc (S36, 1 : 1000, Abcam), PKC β (1 : 1000, Proteintech Group), p-PKC β (T642, 1 : 800, Abcam), PKC δ (1 : 800, Proteintech Group), p-PKC δ (Y311, 1 : 3000, Abcam), cytochrome c (1 : 800, Proteintech Group), GAPDH (1 : 1000, Santa Cruz), and COXIV (1 : 600, Abcam) at 4°C overnight.

Techniques: Activation Assay, Translocation Assay, Expressing, Software, Western Blot, Incubation, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pretreatments with PKC β and PKC δ inhibitors ameliorated CSE-induced mitochondrial damage of airway epithelial cells. Mitochondrial reactive oxygen species (ROS) was observed by confocal microscopy (a), and mitochondrial membrane potential (MMP) was determined by flow cytometry (b). Quantitative analysis showed that pretreatments with LY333531 and rottlerin reduced mitochondrial ROS content (c) and increased the levels of MMP (d) and intracellular ATP (e) in Bease-2b cells stimulated with 7.5% CSE for 24 h. All the results are shown as the mean ± SD from three independent experiments. ∗ P < 0.01 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: The membranes were then incubated with specific antibodies against p66Shc (1 : 1000, Abcam), p-p66Shc (S36, 1 : 1000, Abcam), PKC β (1 : 1000, Proteintech Group), p-PKC β (T642, 1 : 800, Abcam), PKC δ (1 : 800, Proteintech Group), p-PKC δ (Y311, 1 : 3000, Abcam), cytochrome c (1 : 800, Proteintech Group), GAPDH (1 : 1000, Santa Cruz), and COXIV (1 : 600, Abcam) at 4°C overnight.

Techniques: Confocal Microscopy, Membrane, Flow Cytometry, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pretreatments with PKC β and PKC δ inhibitors attenuated CSE-induced airway epithelial cell injury. Cell apoptosis was determined by flow cytometry (a), and apoptosis rates of Beas-2b cells treated with 7.5% CSE and PKC β / δ inhibitors were both significantly decreased compared to the cells treated only with 7.5% CSE (b). Pretreatments with LY333531 and rottlerin significantly upregulated cell viability (c) and downregulated culture supernatant concentration of IL-6 and TNF- α (d) in Beas-2b cells exposed to 7.5% CSE. Data are expressed as the mean ± SD from three independent experiments. ∗ P < 0.01 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: The membranes were then incubated with specific antibodies against p66Shc (1 : 1000, Abcam), p-p66Shc (S36, 1 : 1000, Abcam), PKC β (1 : 1000, Proteintech Group), p-PKC β (T642, 1 : 800, Abcam), PKC δ (1 : 800, Proteintech Group), p-PKC δ (Y311, 1 : 3000, Abcam), cytochrome c (1 : 800, Proteintech Group), GAPDH (1 : 1000, Santa Cruz), and COXIV (1 : 600, Abcam) at 4°C overnight.

Techniques: Flow Cytometry, Concentration Assay, Control