rabbit polyclonal anti pacap igg  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) BiFC assays. Shown were YFP fluorescence intensity re-produced by the transfection of the receptor constructs as indicated. The cells without transfection were used as negative control and the cells transfected with PAC-YFP as positive control. Exogenous NAC (10 nM) decreased the YFP fluorescence intensity produced by PAC-Y/N+PAC-Y/C significantly (*, P<0.01 PAC-Y/N+PAC-Y/C+NAC vs. PAC-Y/N+PAC-Y/C), while the transfection of M+PAC-Y/N+M+PAC-Y/C produced no YFP fluorescence signals. Data were presented as means ± S.E. of three independent experiments. (B) Saturation BRET. Shown were the BRET saturation curves plotted as a ratio of YFP fluorescence to Rlu luminescence that were observed for tagged receptor constructs studied with a fixed amount of donor and increasing amounts of acceptor. PAC-Rluc/PAC-YFP receptor constructs yielded exponential curves that reached asymptotes indicating significant homo-dimerization of PAC1, while M-PAC-Rluc/M-PAC-YFP yielded curves not different from a straight line, indicating that D-PAC1 lost the ability to form dimers. The addition with NAC (10 nM) at 2 h before the BRET signal assay lowered the curves significantly (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were represented as the means ± S.E. of three independent experiments. (C) Static BRET. BRET ratios for CHO cells expressing receptor constructs as indicated. For static BRET, a total of 1.0 µg of DNA per well divided equally among the noted constructs in each condition was utilized. The shaded area represents the nonspecific BRET signal generated between PAC-Rlu and soluble YFP protein, with BRET signals above this area considered to be significant. As shown the BRET ratio in PAC-Rluc/PAC-YFP CHO cells incubated with NAC (10 nM) was significantly lower than that in cells without treatment with NAC (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were presented as the means ± S.E. of three independent experiments. (D) Western blotting analysis with a goat polyclonal IgG against the C-terminus of PAC1 using non-reductive SDS-PAGE. When PAC-YFP expressing cells incubated with exogenous NAC (10 nM), as shown, the band with the molecular weight (about 160 kD) consistent with the molecular weight of the PAC1 dimer was weakened by the presence of NAC (10 nM). All these results showed that NAC was an inhibitor of the dimerization of PAC1, which offered us a tool to analysis the relation of the dimerization of PAC1 with its basal activity.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Fluorescence, Produced, Transfection, Construct, Negative Control, Positive Control, Expressing, Generated, Incubation, Western Blot, SDS Page, Molecular Weight, Activity Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: (A) Western blotting of endogenous PACAP in CHO-K1 and neuro2a cells. As shown CHO-K1 had no detectable endogenous PACAP, while neuro2a cells produced endogenous PACAP. (B) The expression of PAC1-YFP and M-PAC1-YFP detected by immunofluorescence. Shown were immunofluorescence results of PAC1-CHO and M-PAC1-CHO cells cultured in DMEM with 0.5% CS-FBS at 37°C overnight, which indicated that both PAC1 and M-PAC1 trafficked normally to the plasma membrane and 0.5% CS-FBS induced no significant receptors endocytosis. (C) Fluorescence densities assays. Shown were the YFP fluorescence densities in the whole cell lysate detected using the Victor3 1420 multi-label counter with excitation (460±30 nm) and emission (535±30 nm), indicating that the expression levels of PAC1-YFP in CHO cells were equal to those of M-PAC1-YFP. (D) Western blotting assays using reductive SDS-PAGE. Western blotting with a goat polyclonal IgG against the C-terminus of PAC1 in the reductive condition showed that there were similar bands with the molecular weight about 160 kD in PAC1-CHO and M-PAC1-CHO, but not in CHO. (E) The cell viabilities of PAC1-CHO and M-PAC1-CHO cells promoted by PACAP. The data were plotted as the fold changes of the treatment without PACAP (0 nM). After the cells were submitted the addition of PACAP (1–100 nM) in the absence of CS-FBS for 24 h, MTT assays showed that PACAP exerted more significant proliferative effects on M-PAC1-CHO than on PAC1-CHO (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO), indicating that the activation level of PAC1 by PACAP was lower than that of M-PAC1. (F) The intracellular cAMP levels induced by PACAP (1–100 nM) in PAC1-CHO and M-PAC1-CHO cells. After the data were plotted as the fold changes of the treatment with 0 nM PACAP, it was shown that the intracellular cAMP levels in M-PAC1-CHO cells induced by PACAP were significantly higher than the intracellular cAMP levels in PAC1-CHO cells induced by PACAP (*, P<0.01, M-PAC1-CHO vs. PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Western Blot, Produced, Expressing, Immunofluorescence, Cell Culture, Clinical Proteomics, Membrane, Fluorescence, SDS Page, Molecular Weight, Activation Assay

Journal: PLoS ONE

Article Title: Dimer-Dependent Intrinsic/Basal Activity of the Class B G Protein-Coupled Receptor PAC1 Promotes Cellular Anti-Apoptotic Activity through Wnt/β-Catenin Pathways that Are Associated with Dimer Endocytosis

doi: 10.1371/journal.pone.0113913

Figure Lengend Snippet: The fluorescence microscopic observation (A) and the fluorescence density assays (B) of the PAC1-YFP expression induced by Dox (0–100 ng/mL) in Tet-on inducible system. The fluorescence microscopic images showed that the numbers of cells with YFP fluorescence increased following increases in the concentration of Dox (1–100 ng/mL), whereas there was no fluorescence observed without induction by Dox (0 ng/mL). Bar, 20 µm. The YFP fluorescence densities, assayed using the Victor3 1420 multi-label counter, increased with the concentration of Dox (1–100 ng/mL), indicating that the expression levels of PAC1 were controlled by Dox in a concentration-dependent manner. (C) Western blotting of the inducible expression of PAC1-YFP. The western blotting with goat polyclonal IgG against the C-terminus of PAC1 using reductive SDS-PAGE showed the bands corresponding to PAC1-YFP deepened with the increase of Dox (1–100 ng/mL), while no band corresponding to PAC1-YFP was found in the treatment without Dox (0 ng/mL). The remaining cell viabilities (D), the caspase3 activity (E) and the Bcl-2 levels (F) after serum withdrawal in the double-stable Tet-on advanced inducible cells treated with Dox (1–100 ng/mL) were plotted as the fold changes in the data from the cells treated without Dox (0 ng/mL). It was shown that the higher concentrations of Dox induced higher expression levels of PAC1-YFP, which in turn led to the higher anti-apoptotic activity of the cells, including higher remaining cell viability, lower caspase3 activity and higher Bcl-2 level. (G) Top-flash assays. In double-stable Tet-on advanced inducible cells, after the transfection with Top-flash + pRluc or Fop-flash + pRluc, cells were submitted to serum-withdraw induced apoptosis with Dox (1–100 ng/mL) or without Dox for another 24 h. And then cells were lysed and luciferase activities were measured. Relative luciferase activities were expressed as the ratio of TOP-flash/FOP-flash luciferase activity and the data were plotted as fold changes in the data from the cells treated without Dox (0 ng/mL). It was shown that the relative luciferase activities increased following the increase of Dox (1–100 ng/mL), indicating that the higher expression levels of PAC1-YFP induced by higher concentration of Dox resulted into stronger Wnt/β-catenin signals. (H) Western blotting of β-catenin, cyclin D1 and c-myc corresponding to Wnt/β-catenin pathway. After the protein expression levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes of the cells treated without Dox, it was shown that in the cells expressing a range of PAC1-YFP induced by Dox (0–100 ng/mL), β-catenin, cyclin D1 and c-myc levels increased following the increases of the PAC1 levels. All these data suggested the significant positive correlation of the PAC1 levels with the anti-apoptotic activities involved with Wnt/β-catenin signals. The data were represented as the means ± S.E. of three independent experiments.

Article Snippet: To knockdown the endogenous PACAP in neuro2a neuroblastoma cells, cells that were seeded in 6-well plates in DMEM with 10% CS-FBS and cultured to 80% confluence were transfected for 6 h with 4 μg per well PACAP shRNA plasmid (Santa Cruz Biotechnology, USA) using lipofectamine LTX and Opti-MEM medium (Invitrogen, USA), after which the cells were washed and incubated with DMEM and 10% CS-FBS for 24 h. Then, puromycin (PM) (10 μg/mL) was added, and the cells were cultured for another 24 h. The cells were harvested for western blot analysis and were probed with a rabbit polyclonal anti-PACAP IgG (Santa Cruz Biotechnology, USA) that was raised against the C-terminus of PACAP and that recognizes both rodent and human PACAP.

Techniques: Fluorescence, Expressing, Concentration Assay, Western Blot, SDS Page, Activity Assay, Transfection, Luciferase, Control