Journal: Biochimica et biophysica acta

Article Title: A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation.

doi: 10.1016/j.bbamcr.2010.07.001

Figure Lengend Snippet: Fig. 1. Ox-A-induced phosphorylation of protein kinases. (A) Representative Western blot of HEKOx1R cells treated with 5 nM Ox-A for various times (indicated in the figure) and probed with anti-active ERK, anti-PKD1 S916p, anti-PKCα/β T638/641p, anti-PKCδ T505p, and anti-actin (positive control). Ctrl 0 s (first lane) and Ctrl 1 h (last lane) represent control samples treated with vehicle 0 second and 1 hour, respectively. (B) Time profiles of ERK1/2, PKD1 and PKCδ phosphorylation obtained by quantifying the scanned blots from experimental conditions similar to (A) with ImageQuant (n=3). Data are presented as percent of maximal response.

Article Snippet: PhosphoPKC Antibody Sampler Kit, PKD1/PKCμ, Phospho-PKD1/PKCμ Ser744/ 748 and PKD1 Ser916 antibodies were from Cell Signaling (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Positive Control, Control

Journal: Biochimica et biophysica acta

Article Title: A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation.

doi: 10.1016/j.bbamcr.2010.07.001

Figure Lengend Snippet: Fig. 2. Comparison of Ox-A-induced calcium and PKD1 responses. (A) Representative Ca2+ imaging recording with fura-2 AM from single HEKOx1R cell stimulated by increasing nM concentrations of Ox-A indicated in the figure and by 100 μM oxotremorine (M, positive control) at time points indicated by arrows. (B) Representative Western blot of HEKOx1R cells treated with increasing nM concentrations of Ox-A indicated in the figure and probed with anti-PKD1 S916p and anti-actin (positive control). (C) Dose–response curves of [Ca2+]i elevation obtained from Ca2+ imaging recordings with fura-2 AM from cell suspensions of HEKOx1R cells stimulated by increasing concentrations of Ox-A (n=9–40) and of PKD1 activation obtained by quantifying the scanned blots from experimental conditions similar to (B) with ImageQuant (n=2). Data are presented as percent of maximal response±SE.

Article Snippet: PhosphoPKC Antibody Sampler Kit, PKD1/PKCμ, Phospho-PKD1/PKCμ Ser744/ 748 and PKD1 Ser916 antibodies were from Cell Signaling (Danvers, MA, USA).

Techniques: Comparison, Imaging, Positive Control, Western Blot, Activation Assay

Journal: Biochimica et biophysica acta

Article Title: A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation.

doi: 10.1016/j.bbamcr.2010.07.001

Figure Lengend Snippet: Fig. 3. Effect of PKC on Ox-A-induced calcium and PKD1 responses. (A) Representative Western blot of HEKOx1R cells treated with 1 nM Ox-A for 5 minutes in the absence and presence of PKC inhibitor GF-X (1 μM) as indicated in the figure and probed with anti- PKD1 S916p and anti-actin (positive control). (B) Comparison of the effects of GF-X on calcium and PKD1 responses. [Ca2+]i elevations in the absence and presence of 1 μM GF-X were determined in Ca2+ imaging recordings with fura-2 AM from cell suspensions of HEKOx1R cells stimulated by increasing concentrations of Ox-A indicated in the figure (n=2–18). PKD1 activation was obtained by quantifying (ImageQuant) the scanned blots (n≥3) from experimental conditions similar to (A) with increasing concentrations of Ox-A. Responses in the presence of GF-X were compared with the responses without GF-X, and data are presented as percent of control response±SE.

Article Snippet: PhosphoPKC Antibody Sampler Kit, PKD1/PKCμ, Phospho-PKD1/PKCμ Ser744/ 748 and PKD1 Ser916 antibodies were from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Positive Control, Comparison, Imaging, Activation Assay, Control

Journal: Biochimica et biophysica acta

Article Title: A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation.

doi: 10.1016/j.bbamcr.2010.07.001

Figure Lengend Snippet: Fig. 4. Ox-A-induced activation and localization of PKD1 and PKD3. (A) Representative Western blot of HEKOx1R-EGFP-PKD3 cells treated with vehicle, 1, 20, or 50 nM Ox-A for 5 minutes, immunoprecipitated with polyclonal anti-GFP antibody, and probed with anti-active PKD (S744/748P) (n=4) and with anti-GFP (positive control). (B) Representative Western blots of cell membrane fraction of HEKOx1R and HEKOx1R-EGFP-PKD3 cells treated with vehicle, 1 nM or 50 nM Ox-A for 5 minutes and probed with monoclonal anti-GFP in the case of PKD3 (n=2), with anti-PKD1/PKCμ in the case of PKD1 (n=3), and with anti-Ox1R as a positive control (n=3). (C) Epi-fluorescence microscopy images of HEKOx1R-EGFP-PKD3 cells treated with vehicle (control), 1 nM or 50 nM Ox-A for 5 and 30 minutes.

Article Snippet: PhosphoPKC Antibody Sampler Kit, PKD1/PKCμ, Phospho-PKD1/PKCμ Ser744/ 748 and PKD1 Ser916 antibodies were from Cell Signaling (Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Positive Control, Membrane, Microscopy, Control

Journal: Biochimica et biophysica acta

Article Title: A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation.

doi: 10.1016/j.bbamcr.2010.07.001

Figure Lengend Snippet: Fig. 5. Effect of kinase-dead construct of PKD1 (PKD1kd) on Ox-A-induced calcium oscillations. (A) Representative single cell Ca2+ imaging recordings with fura-2 AM from a control cell (left) and a PKD1kd-expressing cell (right) stimulated by 1 nM Ox-A for times indicated by horizontal bars. The cells successfully transfected by PKD1kd were identified based on EGFP fluorescence. (B) Summary of oscillation frequency data obtained from experimental conditions similar to (A). The oscillation frequencies of transiently oscillating PKD1kd-expressing (n=62) and control (nonfluorescent) cells (n=42) from same experiments were calculated as spikes per second (Hz) and compared. The oscillation frequency of control group was set as 100%, and the results are presented as percent frequency±SE.

Article Snippet: PhosphoPKC Antibody Sampler Kit, PKD1/PKCμ, Phospho-PKD1/PKCμ Ser744/ 748 and PKD1 Ser916 antibodies were from Cell Signaling (Danvers, MA, USA).

Techniques: Construct, Imaging, Control, Expressing, Transfection

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Orally Administered Mucolytic Drug l-Carbocisteine Inhibits Angiogenesis and Tumor Growth in Mice.

doi: 10.1124/jpet.115.224816

Figure Lengend Snippet: Fig. 5. L-Carbocisteine inhibits VEGF-induced PLCg/PKC/ERK signaling in HUVECs. (A–D) HUVECs were pretreated with L-carbocisteine and stimulated with VEGF for the indicated periods. Lysates were subjected to SDS-PAGE and the membranes were hybridized with phospho-specific antibodies, after which the membranes were reprobed. Protein levels of p-VEGFR2 (A), p-PLCg (B), p-PKCm (C), and p-MEK1/2 (D) were determined. Quantitative results were obtained by densitometry. Data are presented as mean 6 S.E.M. from three independent experiments. *P , 0.05.

Article Snippet: Anti-phospho-Akt (Ser473), anti-Akt, anti-phospho ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti–phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) (Thr183/Tyr185), anti-SAPK/JNK, anti-MEK1/2, anti–phospho-PLCg (Tyr783), anti-PLCg, anti–phosphoPKCm/PKD (Ser744/748), anti-PKCm/PKD, anti–phospho-VEGFR2 (Tyr1175), anti-VEGFR2, and horseradish peroxidase–conjugated anti-rabbit/ mouse IgG antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Orally Administered Mucolytic Drug l-Carbocisteine Inhibits Angiogenesis and Tumor Growth in Mice.

doi: 10.1124/jpet.115.224816

Figure Lengend Snippet: Fig. 6. L-Carbocisteine attenuated VEGF-induced formation of PLCg/ VEGFR2 complexes. HUVECs were pretreated with L-carbocisteine and stimulated with VEGF for the indicated periods. The cells were harvested and equal aliquots of protein extracts were immunoprecipitated with antibodies against VEGFR2 or PLCg. Immunoprecipitates were subjected to SDS-PAGE and blotted with antibodies against PLCg or VEGFR2 as indicated. Total cell extracts were prepared and subjected to SDS-PAGE for detection of VEGFR2 and PLCg. The blot was reprobed with beta-actin antibodies as a loading control. Data are presented as mean 6 S.E.M. from three independent experiments. *P , 0.05.

Article Snippet: Anti-phospho-Akt (Ser473), anti-Akt, anti-phospho ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti–phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) (Thr183/Tyr185), anti-SAPK/JNK, anti-MEK1/2, anti–phospho-PLCg (Tyr783), anti-PLCg, anti–phosphoPKCm/PKD (Ser744/748), anti-PKCm/PKD, anti–phospho-VEGFR2 (Tyr1175), anti-VEGFR2, and horseradish peroxidase–conjugated anti-rabbit/ mouse IgG antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Immunoprecipitation, SDS Page, Control