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1.
Figure 4

Figure 4. RT-PCR analysis of rat DRG. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

Rat DRG express mRNA for PKD1, PKD2 and PKD3. Lanes on gels are as follows: lane 1, rat PKD1 control (no reverse transcriptase); lane 2, rat PKD1 product size 448 bp; lane 3, rat PKD2 control; lane 4, rat PKD2 product size 479 bp; lane 5, rat PKD3 control; lane 6, rat PKD3 product size 445 bp; lane 7, blank; lane 8, ladder.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
2.
Figure 6

Figure 6. Characterization of the distribution of PKD3 in histological sections of rat DRG. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

PKD3-LI was detected in histological sections of rat DRG and co-localized with neuronal markers. PKD3-LI (A,D,G,J,M; green) was detected at the plasma membrane (yellow arrow heads) and in the cytosol (asterisks) of large, medium and small diameter neurons. In some neurons PKD3-LI was co-localized with neuronal markers (magenta): CGRP (B,C), TRPV1 (E,F), PAR2 (H,I), IB4 (K,L) and N52 (N,O) (white arrows). n = 3 experiments. Scale bar = 25 μm.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
3.
Figure 5

Figure 5. Characterization of the distribution of PKD1 and PKD2 in histological sections of rat DRG. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

PKD1/2-LI was detected in histological sections of rat DRG using an antibody that recognizes both PKD1 and PKD2 and co-localized with neuronal markers. PKD1/2-LI (A,D,G,J,M; green) was detected at the plasma membrane (yellow arrow heads) and in the cytosol (asterisks) of large, medium and small diameter neurons. In some neurons, PKD1/2-LI was co-localized with neuronal markers (magenta): CGRP (B,C), TRPV1 (E,F), PAR2 (H,I), IB4 (K,L) and N52 (N,O) (white arrows). n = 3 experiments. Scale bar = 25 μm.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
4.
Figure 7

Figure 7. Characterization of the distribution of PKD/2 and PKD3 in histological sections of mouse DRG. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

PKD1/2 -LI (A–F) and PKD3-LI (G–L) were detected in histological section of mouse DRG and co-localized with the neuronal markers CGRP and IB4. PKD1/2-LI (A,D; green) and PKD3-LI (G–J; green) were detected at the plasma membrane (yellow arrow heads) and in the cytosol (asterisks) of large, medium and small diameter neurons. In some neurons PKD1/2 was co-localized with neuronal markers (magenta): CGRP (B,C) and IB4 (E,F). PKD3 was co-localized with CGRP (H,I) and IB4 (K,L) (white arrows head). n = 3 experiments. Scale bar = 25 μm.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
5.
Figure 3

Figure 3. Stimulation of PAR2 induced phosphorylation of PKD-GFP in HEK293 cells determined by Western blotting. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

HEK293 cells expressing PKD1- (A, B), PKD2- (C, D) or PKD3-GFP (E, F) were incubated with PAR2-AP (100 μM) for 0–30 min, PMA (100 nM) or PAR2-RP (100 μM) for 5 min. Kinase activation was monitored by Western blotting (A, C, E) and densitometry (B, D, F) measuring the level of pSer744/748 normalized to GFP signal. In non-treated cells (NT), a p-S744/748-LI was undetectable. Treatment with PAR2-AP and PMA (used as positive control) but not with PAR2-RP (used as negative control) increased levels of pSer744/748, suggesting activation. n ≥ 3 experiments. *p < 0.05 versus NT.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
6.
Figure 2

Figure 2. PAR2-induced activation of PKD-GFP is mediated by PKCε. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

HEK293 cells expressing PKD1- (A), PKD2- (B) or PKD3-GFP (C) were incubated with PAR2-AP (100 μM) for 5 and 15 min in the presence of PKCε inhibitor peptide (PKCεI) or the inactive scrambled sequence (ScPKCεI) (10 μM, 15 min). Kinase activation was monitored by measuring the level of pSer744/748-LI normalized to GFP signal. Treatment with PAR2-AP increased the levels of pSer744/748-LI compared to non-treated cells (NT), suggesting activation of PKDs. Pre-treatment with PKCεI reduced the increase of phosphorylated PKD1- and PKD2-GFP induced when PAR2-AP was applied for 5 min, and of phosphorylated PKD3-GFP induced when PAR2-AP was applied for 5 or 15 min. n=3 experiments. n=80-145 cells analyzed per group. *p < 0.05 versus ScPKCεI-treated.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
7.
Figure 9

Figure 9. Intraplantar injection of PAR2-AP activates PKD in lumbar DRG. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

Test compounds were injected into the mouse paw. After 30 min, lumbar DRG (L4-L6) were collected and PKD activation monitored by immunostaining (A–C) and quantified by densitometry (D) using pSer744/748 antibody for phosphorylated PKD in large (diameter > 30 μm) and medium/small size (diameter ≤ 30 μm) neurons of histological sections. PAR2-AP (10 μg/10 μl/ paw) increased levels of pSer744/748-LI (C,D) in lumbar medium/small size neurons but not in large neurons, compared to mice treated with saline (A,D) or PAR2-RP (B,D). Scale bar = 25 μm. * p <0.05 compared to saline or PAR2-RP. n ≥ 3 experiments. Numbers above bars = number of neurons analyzed per group.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
8.
Figure 1

Figure 1. Stimulation of PAR2 induced translocation and phosphorylation of PKD-GFP in HEK293 cells determined by immunostaining. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

HEK293 cells expressing PKD1- (A1-I1, A4-C4), PKD2- (A2-I2, D4-F4) or PKD3-GFP (A3-I3, G4-I4) were incubated with PAR2-AP (100 μM) for 0, 1 or 5 min or with PMA (100 nM) for 5 min. PKDs were localized by immunostaining using GFP (green) and activation was monitored with a pSer744/748 antibody for phosphorylated kinases (magenta). In non-treated cells (0 min), PKD1- (A1-C1), PKD2- (A2-C2) and PKD3-GFP (A3-C3) were localized in the cytosol (white arrows), and PKD3-GFP was also localized in the nucleus (asterisks). pSer744/748-LI was undetectable. After incubation with PAR2-AP for 1 min, GFP and pSer744/748 were detected at the plasma membrane (PKD1-GFP: D1-F1; PKD2-GFP: D2-F2; PKD3-GFP: D3-F3; arrow heads). At 5 min, GFP and the corresponding pSer744/748-LI returned to the cytosol (PKD1-GFP: G1-I1; PKD2-GFP: G2-I2; PKD3-GFP: G3-I3; white arrows). Note that some PKD3-GFP and the corresponding pSer744/748-LI were still detected at the plasma membrane. PMA induced translocation of PKD1-GFP (A4-C4), PKD2-GFP (D4-F4), and PKD3-GFP (G4-I4) to the plasma membrane and the appearance of pSer744/748-LI (arrow heads). n ≥ 3 experiments. Scale bar = 20 μm.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.
9.
Figure 8

Figure 8. Stimulation of PAR2 induced activation of PKD in rat DRG in culture. From: Protein Kinase D Isoforms Are Expressed In Rat and Mouse Primary Sensory Neurons and Are activated by Agonists Of Protease-Activated Receptor 2.

Rat DRG in culture were stimulated with different test compounds and PKD activation was monitored by immunostaining (A–I) and quantified by densitometry (J) using pSer744/748 antibody for phosphorylated kinases. Treatments: A, vehicle (veh); B, PDBu, 100 nM, 5 min; C, inflammatory mixture (Inf. Mix: bradykinin (1 μM), SP (1 μM), 5-hydroxytryptamine (10 μM) and histamine (10 μM)) for 1 min; D, inflammatory mixture, 5 min; E, PAR2-RP, 100 μM, 5 min; F, PAR2-AP, 100 μM, 1 min; G, PAR2-AP, 100 μM, 5 min. H, PAR2-AP, 100 μM, 15 min; I, no treatment and no primary antibody. Note that PDBu and the inflammatory mixture increased levels of pSer744/748-LI compared to cells treated with vehicle, suggesting PKD activation. PAR2-AP increased levels of pSer744/748-LI compared to cells treated with PAR2-RP (J). White asterisk indicates increased pSer744/748-LI. No pSer744/748-LI was detected when primary antibody was omitted. For the quantification, pSer744/748-LI intensity (pixels) was normalized to cell area (μm). Scale bar = 25 μm. *, ** p <0.05 compared to vehicle (*) or to PAR2-RP (**). n = 3 experiments. n ≥ 3 experiments. Numbers above bars = number of cells analyzed per group.

Silvia Amadesi, et al. J Comp Neurol. ;516(2):141-156.

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