Journal: Journal of Biological Chemistry

Article Title: PKCδ Mediates Testosterone-induced Increases in Coronary Smooth Muscle Cav1.2

doi: 10.1074/jbc.m509147200

Figure Lengend Snippet: FIGURE 5. Specific down-regulation of coronary PKC protein siRNA. Representative immunoblots showing PKC siRNA-treated and untreated CSMC whole cell homoge- nates probed with anti-PKC, -, -, and - antibodies. For the evaluation of protein loading, the blots were stripped and reprobed for -actin. PKC siRNA successfully knocked down PKC protein levels without altering the expression of PKC , -, and - in CSMC, providing evidence for specific down-regulation of PKC protein levels. Autora- diograms are representative of four independent experiments for each isoform.

Article Snippet: Briefly, 5 l of Lipofectamine 2000 (Invitrogen) and 20 pM PKC siRNA or control siRNA pool (Santa Cruz Biotechnology) were diluted in 250 l of serum-free phenol red-free Dulbecco’s modified Eagle’s medium and incubated at room temperature for not more than 5 min.

Techniques: Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: PKCδ Mediates Testosterone-induced Increases in Coronary Smooth Muscle Cav1.2

doi: 10.1074/jbc.m509147200

Figure Lengend Snippet: FIGURE 6. PKC siRNA inhibits up-regulation of coronary Cav1.2 protein by testos- terone. Densitometric analysis of the effect of PKC siRNA on testosterone-induced up-regulation of Cav1.2 protein (n 6). PKC siRNA-treated ( siRNA) and untreated ( siRNA) CSMC were incubated for 18 h in the absence (Control) or presence (T; 100 nM) of testosterone for 18 h. Each band was normalized to the average control value obtained from the same blot. Top, representative immunoblot showing homogenates probed with anti-Cav1.2 antibody from corresponding groups. Cav1.2-positive band appeared at 220 kDa. PKC siRNA inhibited testosterone up-regulation of Cav1.2 pro- tein levels. Values are mean S.E. *, p 0.05 versus all.

Article Snippet: Briefly, 5 l of Lipofectamine 2000 (Invitrogen) and 20 pM PKC siRNA or control siRNA pool (Santa Cruz Biotechnology) were diluted in 250 l of serum-free phenol red-free Dulbecco’s modified Eagle’s medium and incubated at room temperature for not more than 5 min.

Techniques: Incubation, Control, Western Blot

Journal: Molecular medicine reports

Article Title: Oxidized-low density lipoprotein accumulates cholesterol esters via the PKCα-adipophilin-ACAT1 pathway in RAW264.7 cells.

doi: 10.3892/mmr.2015.3864

Figure Lengend Snippet: Figure 1. Ox‑LDL increases the phosphorylation of PKCα, and the expression levels of adipophilin and ACAT1, and increases lipid droplet accumulation. (A) RAW264.7 cells were incubated with either 50 mg/ml Ox‑LDL for 24 h or with 10% BSA for 24 h. The mRNA expression levels of PKCα, adipophilin and ACAT1 were analyzed using reverse transcription‑quantitative polymerase chain reaction and normalized to GAPDH transcripts. (B‑D) Expression levels of PKCα, p‑PKCα, edipophilin and ACAT1 in whole cell lysates from RAW264.7 cells treated for 24 h with 50 mg/ml Ox‑LDL, analyzed using western blot analysis and densitometry. (E) RAW264.7 cells were incubated with either 50 mg/ml Ox‑LDL for 24 h or 10% BSA for 24 h. The cells were then stained with oil red O. Intracellular lipid droplets are stained red and nuclei are stained blue (magnification, x100). Ox‑LDL, oxidized low density lipoprotein; con, control; PKC, protein kinase C; p‑, phosphorylated; ATAC1, adipophilin and acyl‑coenzymeA:cholesterol acyltransferse 1.

Article Snippet: Transfection of small interfering (si)RNA. siRNA targeting PKCα, adipophilin and ACAT1 was purchased from Santa Cruz Biotechnology, Inc. A control siRNA, specific for red fluorescent protein (CCACTACCTGAGCACCCAG) was used as a negative control.

Techniques: Phospho-proteomics, Expressing, Incubation, Polymerase Chain Reaction, Western Blot, Staining, Control

Journal: Molecular medicine reports

Article Title: Oxidized-low density lipoprotein accumulates cholesterol esters via the PKCα-adipophilin-ACAT1 pathway in RAW264.7 cells.

doi: 10.3892/mmr.2015.3864

Figure Lengend Snippet: Figure 2. Ox‑LDL‑induced expression of adipophilin and ACAT1 by PKCα. (A) RAW264.7 cells were pre‑incubated with PKCα siRNA or NA for 24 h followed by 50 mg/ml ox‑LDL for 24 h. mRNA levels of adipophilin and ACAT1 were determined using reverse transcription‑quantitative polymerase chain reaction and normalized to GAPDH. (B‑D) RAW264.7 cells were pre‑incubated with 10 µM PKCα siRNA for 24 h followed by 50 mg/ml ox‑LDL for 24 h. Cell proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with polyclonal anti‑adipophlin and anti‑ACAT1 antibodies. All data are expressed as the mean ± standard deviation of three independent experiments, each performed in triplicate. *P<0.05, vs. ox‑LDL group; #P<0.05, vs. ox‑LDL+NA. Ox‑LDL, oxidized low density lipoprotein; siRNA, small interfering RNA; NA, scramble siRNA; con, control; PKC, protein kinase C; p‑, phosphorylated; ATAC1, adipophilin and acyl‑coenzymeA:cholesterol acyltransferse 1.

Article Snippet: Transfection of small interfering (si)RNA. siRNA targeting PKCα, adipophilin and ACAT1 was purchased from Santa Cruz Biotechnology, Inc. A control siRNA, specific for red fluorescent protein (CCACTACCTGAGCACCCAG) was used as a negative control.

Techniques: Expressing, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Standard Deviation, Small Interfering RNA, Control

Journal: Molecular medicine reports

Article Title: Oxidized-low density lipoprotein accumulates cholesterol esters via the PKCα-adipophilin-ACAT1 pathway in RAW264.7 cells.

doi: 10.3892/mmr.2015.3864

Figure Lengend Snippet: Figure 4. Adipophilin siRNA attenuates oxLDL‑mediated expression of ACAT1. Cells were transfected with either adipophilin siRNA or NA for 24 h, prior to treatment with ox‑LDL (50 mg/ml). (A) mRNA expression of ACAT1 was determined using reverse transcription‑quantitative polymerase chain reaction. (B) Western blot analysis and (C) densitometric quantification of the protein levels of ACAT1 in the protein extracts from each group. The results are expressed as the mean ± standard deviation from three inde pendent experiments, each performed in triplicate. ﹡P<0.05, vs. ox‑LDL; #P<0.05, vs. ox‑LDL+NA. Ox‑LDL, oxidized low density lipoprotein; siRNA, small interfering RNA; NA, scramble siRNA; con, control; ATAC1, adipophilin and acyl‑coenzymeA:cholesterol acyltransferse 1.

Article Snippet: Transfection of small interfering (si)RNA. siRNA targeting PKCα, adipophilin and ACAT1 was purchased from Santa Cruz Biotechnology, Inc. A control siRNA, specific for red fluorescent protein (CCACTACCTGAGCACCCAG) was used as a negative control.

Techniques: Expressing, Transfection, Polymerase Chain Reaction, Western Blot, Standard Deviation, Small Interfering RNA, Control

Journal: Molecular medicine reports

Article Title: Oxidized-low density lipoprotein accumulates cholesterol esters via the PKCα-adipophilin-ACAT1 pathway in RAW264.7 cells.

doi: 10.3892/mmr.2015.3864

Figure Lengend Snippet: Figure 5. Adipophilin siRNA attenuates ox‑LDL‑induced accumulation of intracellular lipid droplets. RAW264.7 cells were pre‑incubated with adi pophilin siRNA for 24 h, prior to incubation with 50 mg/ml ox‑LDL. The cells were then stained with oil red O. Intracellular lipid droplets are stained red and nuclei are stained blue (magnification, x100). Ox‑LDL, oxidized low density lipoprotein; siRNA, small interfering RNA; NA, scramble siRNA; con, control.

Article Snippet: Transfection of small interfering (si)RNA. siRNA targeting PKCα, adipophilin and ACAT1 was purchased from Santa Cruz Biotechnology, Inc. A control siRNA, specific for red fluorescent protein (CCACTACCTGAGCACCCAG) was used as a negative control.

Techniques: Incubation, Staining, Small Interfering RNA, Control

Journal: Molecular medicine reports

Article Title: Oxidized-low density lipoprotein accumulates cholesterol esters via the PKCα-adipophilin-ACAT1 pathway in RAW264.7 cells.

doi: 10.3892/mmr.2015.3864

Figure Lengend Snippet: Figure 3. Ox‑LDL‑induced intracellular lipid droplet accumulation is mediated by PKCα signaling. RAW264.7 cells were pre‑incubated with PKCα siRNA for 24 h, and incubated with 50 mg/ml ox‑LDL The cells were then stained with oil red O. Intracellular lipid droplets are stained red and nuclei are stained blue (magnification, x100). Ox‑LDL, oxidized low density lipoprotein; siRNA, small interfering RNA; NA, scramble siRNA; con, control; PKC, protein kinase C.

Article Snippet: Transfection of small interfering (si)RNA. siRNA targeting PKCα, adipophilin and ACAT1 was purchased from Santa Cruz Biotechnology, Inc. A control siRNA, specific for red fluorescent protein (CCACTACCTGAGCACCCAG) was used as a negative control.

Techniques: Incubation, Staining, Small Interfering RNA, Control

Journal:

Article Title: Regulation of (Pro)Renin Receptor Expression by Glucose-Induced Mitogen-Activated Protein Kinase, Nuclear Factor-?B, and Activator Protein-1 Signaling Pathways

doi: 10.1210/en.2009-1368

Figure Lengend Snippet: Effect of glucose and different intracellular signaling pathways on PRR expression. A, Inhibition of PKC with Chelerythrine (Che; 5 μm) and Rottlerin (Ro; 5 μm). B, Inhibition of MAPKs with U0126 (10 μm) and SP600125 (20 μm). C, Inhibition of Raf-1 with GW5074 (10 nm). D, Inhibition of AP-1 with Curcumin (100 μm). E, Inhibition of NF-κB with Quinazoline (NF-κBi) (10 μm). Control, 5 mm d-glucose+25 mm l-glucose; glucose, 30 mm d-glucose. All the results represent the average of three independent experiments and each experiment was repeated at least three times.

Article Snippet: Primary antibodies against ATP6AP2 , TATA binding protein (Abcam, Cambridge, MA), PKCα, PKCβI, PKCγ, PKCδ, Raf-1, phospho-Raf-1 (Y340/341), NF-κB p65, NF-κB p50, NF-κB p52, Jun B, Jun D, c-Jun, phospho-c-Jun (S63), c-Fos, Sp1, Sp3 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-NF-κB p65 (S536) (Cell Signaling Technology, Danvers, MA), ERK1/2, phospho-ERK1/2 (T185/Y187), JNK, and phospho-JNK (T183/Y185) (Invitrogen) were used in this study.

Techniques: Protein-Protein interactions, Expressing, Inhibition, Control

Journal:

Article Title: Regulation of (Pro)Renin Receptor Expression by Glucose-Induced Mitogen-Activated Protein Kinase, Nuclear Factor-?B, and Activator Protein-1 Signaling Pathways

doi: 10.1210/en.2009-1368

Figure Lengend Snippet: PKC isomer proteins and their translocation, phosphorylation of intracellular signal proteins in response to high glucose alone, and combined with different kinases inhibitors in RMCs. Panel A, PKC isomers in membranous (M), cytosolic (C), and nuclear (N) compartments. Loading control (LC), β-Actin for membranous and cytosolic fractions and TATA binding protein (TBP) for nuclear fraction. Panels B–D, Phosphorylation of intracellular signal proteins in response to high glucose alone and combined with different kinases inhibitors. After 2 wk of glucose exposure and 12 h of serum starvation, cells were exposed to inhibitors for 15, 30, and 60 min. Panel B, PKC inhibition and phosphorylation of Raf-1, ERK1/2, JNK, c-Jun, and NF-κB p65. Panel C, Raf-1 inhibition and phosphorylation of ERK1/2, JNK, c-Jun, and NF-κB p65. Panel D, MEK1/2 and JNK inhibition and phosphorylation of NF-κB p65 and c-Jun. Control, 5 mm d-glucose+25 mm l-glucose; glucose, 30 mm d-glucose. The results are representative of three independent experiments.

Article Snippet: Primary antibodies against ATP6AP2 , TATA binding protein (Abcam, Cambridge, MA), PKCα, PKCβI, PKCγ, PKCδ, Raf-1, phospho-Raf-1 (Y340/341), NF-κB p65, NF-κB p50, NF-κB p52, Jun B, Jun D, c-Jun, phospho-c-Jun (S63), c-Fos, Sp1, Sp3 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-NF-κB p65 (S536) (Cell Signaling Technology, Danvers, MA), ERK1/2, phospho-ERK1/2 (T185/Y187), JNK, and phospho-JNK (T183/Y185) (Invitrogen) were used in this study.

Techniques: Translocation Assay, Phospho-proteomics, Control, Binding Assay, Inhibition

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: The KIBRA and p -PKMζ are activated in contralateral ACC neurons after CPTP. (A-F) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, and PKMζ levels in contralateral ACC at different time points in Thoracotomy pain (A-C) or Thoracotomy no pain (D-F), * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs Sham group; the data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4∼7 rats in each group. (G-H) The photographs of the double immunofluorescence staining show that KIBRA and p -PKMζ are only colocalized with neuron markers (NeuN) but not with astrocyte marker (GFAP) and microglia marker (Iba1) in the Sham and CPTP groups. The white arrowheads indicate the co-immunostaining. Scale bar, 100 μm. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter molecule 1; KIBRA, kidney and brain-expressed protein; NeuN, neuron-specific nuclear protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Double Immunofluorescence Staining, Marker, Immunostaining, Binding Assay

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Knockdown of KIBRA in contralateral ACC prevents the upregulation of KIBRA, p -PKMζ/GluR1 signaling pathways, and neuroinflammation in CPTP rats. (A-F) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, TNF-α, and IL-1β levels in ACC analyzed 21 days after the sham operation, thoracotomy, or KIBRA − + thoracotomy. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4∼8 rats in each group. ACC, anterior cingulate gyrus; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Knockdown, Protein-Protein interactions

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: Overexpression of KIBRA in ACC causes allodynia and activates p -PKMζ/GluR1 signaling pathways and neuroinflammation. (A) Experiment designs are shown. (B) Mechanical hyperalgesia ratio on 7, 14, and 21 days after thoracotomy or KIBRA overexpression. **** P < 0.0001 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points for pain rats after injection of rAAV-CMV-wwc1-3xFLAG-WPREs or thoracotomy, * P < 0.05, *** P < 0.001, and **** P < 0.0001 vs Sham group, &&&& P < 0.00001 vs KIBRA + pain group, the data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 12 in KIBRA + pain, n = 8 in KIBRA + no pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed 21 days in Sham, KIBRA + pain, and KIBRA + no pain rats. The data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test, n = 4 in each group. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression, Protein-Protein interactions, Injection

Journal: Pain

Article Title: Kidney and brain-expressed protein upregulation in the anterior cingulate cortex mediates chronic post-thoracotomy pain by the phospho-protein kinase Mζ/glutamate receptor 1 signaling pathway and neuroinflammation in male rats

doi: 10.1097/j.pain.0000000000003849

Figure Lengend Snippet: ACC overexpression KIBRA combined with thoracotomy induced CPTP in all rats. (A) Experiment designs are shown. (B) Incidence of pain on 7, 14, and 21 days after thoracotomy in KIBRA overexpression rats. ** P < 0.01 vs Thoracotomy pain group; the data were analyzed by Fisher exact test. (C and D) The threshold% and mechanical pain threshold at different time points in KIBRA + no pain + Thoracotomy pain, Thoracotomy pain, or Sham group, **** P < 0.0001 vs Sham group, & P < 0.05 vs Thoracotomy pain group. The data were analyzed by 2-way ANOVA followed by Tukey multiple comparisons test, n = 8 in Sham, n = 13 in Thoracotomy pain, n = 14 in KIBRA + no pain + Thoracotomy pain group. (E-J) Representative blots and columnar statistical charts show KIBRA, p -PKMζ, PKMζ, GluR1, TNF-α, and IL-1β levels in ACC analyzed on POD21 in Sham, Thoracotomy pain, or KIBRA + no pain + Thoracotomy pain rats, n = 4 rats in each group. The data were analyzed by 1-way ANOVA followed by Tukey post hoc test. ACC, anterior cingulate gyrus; ANOVA, analysis of variance; CPTP, chronic post-thoracotomy pain; GluR1, glutamate receptor 1; IL-1β, interleukin-1β; KIBRA, kidney and brain-expressed protein; PKMζ, protein kinase Mζ; p -PKMζ, phosphorylated protein kinase Mζ; TNF-α, tumor necrosis factor-α.

Article Snippet: After using the Pierce Bicinchoninic Acid Assay kit to measure the protein concentration, 30 μg protein was separated with SDS-PAGE gels (Bio-Rad, Hercules, CA) and immunoblotted with antibodies against KIBRA (bs-11570R; Bioss, Woburn, MA), GluR1 (67642-1-lg; Proteintech, Rosemont, IL), p -PKMζ (AF3404; Affinity, Cincinnati, OH), PKMζ (sc-17781; Santa Cruz, Dallas, TX), β-actin (EM21002; HUABIO, Hangzhou, China), tumor necrosis factor-α (TNF-α, BS-1857; Bioworld, Dublin, OH), and interleukin-1β (IL-1β, ab9722; Abcam, Cambridge, United Kingdom).

Techniques: Over Expression

Journal: Acta Histochemica et Cytochemica

Article Title: Protective Effect of Ebselen on Ischemia-reperfusion Injury in Epigastric Skin Flaps in Rats

doi: 10.1267/ahc.22-00062

Figure Lengend Snippet: Immunohistochemical analysis for phosphorylated PKCδ and PKCδ in I/R-induced skin flap injury. ( A ) Skin flap from vehicle administration and ebselen administration groups harvested at 1 hr, 6 hr, 12 hr, and 7 days after I/R were stained with anti-PKCδ antibodies and anti-phosphorylated PKCδ antibodies. Representative sections are presented (bar = 100 μm). Inserts are enlarged views (bar = 200 μm). ( B ) Comparison of intensity score for phosphorylated PKCδ in each group. ( C ) Comparison of intensity score for PKCδ in each group. Each bar represents mean ± SD for six rats. *p < 0.05.

Article Snippet: Then, the sections were reacted with the primary antibodies, rabbit IgG against PKCα, PKCδ (1:100; Santa Cruz) in 1% BSA/PBS overnight at 4°C.

Techniques: Immunohistochemical staining, Staining, Comparison