Journal: The Journal of Biological Chemistry

Article Title: Developmental Regulation of Synthesis and Dimerization of the Amyloidogenic Protease Inhibitor Cystatin C in the Hematopoietic System *

doi: 10.1074/jbc.M113.538041

Figure Lengend Snippet: Contribution of bone marrow-derived cells to the serum levels of Cst C. A–C, wild-type (CstC+/+) or cystatin C knock-out (CstC−/−) mice were reconstituted with wild-type or cystatin C-deficient BM as indicated by the arrows. The total splenocytes (A) or blood (B and C) of the reconstituted mice were examined for cystatin C contents by Western blot (A) or ELISA (B and C). Blood samples from cystatin C knock-out mice were included as negative control. ELISA results are presented as average values of duplicates from eight (B) or three (C) mice per group. Error bars represent S.E., and n.d. indicates not detected. **, p < 0.01. D, total cell lysates from purified splenic cell populations as indicated, or DC or macrophages generated in culture from BM precursors (BM-MΦ, BM-DC) were run in SDS-PAGE and their cystatin C contents were examined by Western blot. The same membrane was probed with anti-Actin antibody as loading control. E, purified cell populations as indicated were metabolically labeled and newly synthesized cystatin C was immunoprecipitated from cell lysates (lanes C) or the culture supernatant (lanes S), visualized by autoradiography and quantitated in a phosphorimager. All results are representative of two (MΦ) or multiple (DC) independent experiments performed.

Article Snippet: Confirmation of transplantation was performed by FACS analysis of splenocytes or blood cells measuring the percentage of donor cells. . ELISA The concentration of cystatin C in serum was determined using the mouse DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. . Radioactive Pulse-Chase Analysis Pulse-chase experiments were carried out as previously described ( 24 ).

Techniques: Derivative Assay, Knock-Out, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control, Purification, Generated, SDS Page, Membrane, Metabolic Labelling, Labeling, Synthesized, Immunoprecipitation, Autoradiography

Journal: The Journal of Biological Chemistry

Article Title: Developmental Regulation of Synthesis and Dimerization of the Amyloidogenic Protease Inhibitor Cystatin C in the Hematopoietic System *

doi: 10.1074/jbc.M113.538041

Figure Lengend Snippet: Primary DC contain inactive cystatin C dimers. A, lysates of freshly isolated splenic CD8+ DC were incubated with immobilized, carboxymethylated (proteolytically inactive) papain to precipitate active cystatin C (I). A saturating amount of papain was used as confirmed by the lack of additional cystatin C recovered during a second round of precipitation with the same reagent (II). Remaining (inactive) cystatin C was retrieved by immunoprecipitation using an anti-cystatin C rabbit serum (III). Both the papain-reactive and inactive fractions of cystatin C were run in SDS-PAGE and visualized by Western blot. B, lysate of DC as above was either left untreated (control, upper gel) or incubated with carboxymethylated papain to deplete the active cystatin C (Post-papain, lower gel) before gel filtration chromatography. Cystatin C was retrieved from the fractions by immunoprecipitation using an anti-cystatin C rabbit serum. The immunoprecipitates were separated by 11.5% SDS-PAGE, and cystatin C was visualized by Western blot. The results were quantitated by densitometry (bottom graph). C, total cell lysates (TCL) of CD8+ DC were mixed with sample buffer lacking (−) or containing (+) 2-mercaptoethanol (2ME) as a reducing agent, fractionated by SDS-PAGE and analyzed by Western blot to detect cystatin C. All results are representative of two to three independent experiments.

Article Snippet: Confirmation of transplantation was performed by FACS analysis of splenocytes or blood cells measuring the percentage of donor cells. . ELISA The concentration of cystatin C in serum was determined using the mouse DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. . Radioactive Pulse-Chase Analysis Pulse-chase experiments were carried out as previously described ( 24 ).

Techniques: Isolation, Incubation, Immunoprecipitation, SDS Page, Western Blot, Filtration, Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Developmental Regulation of Synthesis and Dimerization of the Amyloidogenic Protease Inhibitor Cystatin C in the Hematopoietic System *

doi: 10.1074/jbc.M113.538041

Figure Lengend Snippet: Cystatin C dimerizes in the ER, but is mostly secreted as monomers. A, newly synthesized proteins from splenic CD8+ DC were metabolically labeled with [35S]Met/Cys. The cells were equally divided into pulsed and chased samples. The pulsed samples were lysed immediately, whereas the chased samples were incubated for the indicated times before lysis. Cystatin C monomer (mon) and dimer (dim) from either cell lysates (intracellular) or cell culture supernatants (S/N) were retrieved by precipitation with immobilized papain and then by immunoprecipitation with anti-cystatin C serum, respectively. The immunoprecipitates were fractionated by SDS-PAGE, and revealed by autoradiography. B, freshly isolated CD8+ DC were treated as in A. Cell lysates from pulsed samples (top gel) or culture supernatants after a 120-min chase (bottom gel) were fractionated by gel-filtration chromatography. Cystatin C was retrieved from the fractions by immunoprecipitation using an anti-cystatin C rabbit serum. The immunoprecipitates were separated by 11.5% SDS-PAGE and cystatin C was visualized by autoradiography. The band intensities were quantitated in a phosphorimager (bottom graph). C, newly synthesized proteins from splenic CD8+ DC were metabolically labeled with 35S in the presence of the ER-Golgi protein transport inhibitor BFA, and cystatin C was retrieved as in A (pulse). D, Splenic CD8+ DC were metabolically labeled in the presence of BFA and chased in the presence of BFA plus, where indicated, the proteasome inhibitor Lactacystin (Lact). Papain-reactive monomers and non-reactive dimers were immunoprecipitated and run in SDS-PAGE as in A. The amount of radioactive cystatin C in each lane was quantitated in a phosphorimager. E, as in D, but cells were pulsed without drugs and chased in the presence or absence of leupeptin. All results are representative of three or more independent experiments performed.

Article Snippet: Confirmation of transplantation was performed by FACS analysis of splenocytes or blood cells measuring the percentage of donor cells. . ELISA The concentration of cystatin C in serum was determined using the mouse DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. . Radioactive Pulse-Chase Analysis Pulse-chase experiments were carried out as previously described ( 24 ).

Techniques: Synthesized, Metabolic Labelling, Labeling, Incubation, Lysis, Cell Culture, Immunoprecipitation, SDS Page, Autoradiography, Isolation, Filtration, Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Developmental Regulation of Synthesis and Dimerization of the Amyloidogenic Protease Inhibitor Cystatin C in the Hematopoietic System *

doi: 10.1074/jbc.M113.538041

Figure Lengend Snippet: Intracellular ROS levels correlate with cystatin C dimerization. A, macrophages (MΦ) or DC generated in vitro from BM precursors were lysed and cystatin C was precipitated with carboxymethylated papain followed by immunoprecipitation with anti-cystatin C serum as described in the legend to Fig. 2A. The samples were fractionated in SDS-PAGE and cystatin C detected by Western blot. B, cystatin C monomer and dimer synthesis and secretion by MΦ was analyzed as described for DC in the legend to Fig. 3A. C, macrophages and DC were loaded with the cell-permeable, redox-sensitive dye CM-H2DCFDA (5- (and 6-)chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate), and their intracellular ROS levels were examined by flow cytometry. The graph shows the mean fluorescence intensity (MFI) in the FITC channel. The data are presented as the mean ± S.E. of three independent experiments performed. D, the intracellular ROS levels in CD8+ DC freshly isolated from spleens (immature), or in their counterparts cultured overnight (mature), were measured by FACS. E, lysates of purified immature (left) and mature (right) CD8+ DC were fractionated by gel filtration chromatography. Cystatin C was immunoprecipitated from the fractions, run in SDS-PAGE, visualized by Western blot, and quantitated by densitometry (bottom graphs). F, lysates of mature CD8+ DC were analyzed as described in the legend to Fig. 2A to retrieve cystatin C monomer (papain-binding) and dimer (sequentially immunoprecipitated with anti-cystatin C serum). G, mature CD8+ DC were metabolically labeled/chased, and processed as described in the legend to Fig. 3B to measure cystatin C monomer and dimer synthesis and secretion. All results are representative of two to three independent experiments performed.

Article Snippet: Confirmation of transplantation was performed by FACS analysis of splenocytes or blood cells measuring the percentage of donor cells. . ELISA The concentration of cystatin C in serum was determined using the mouse DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. . Radioactive Pulse-Chase Analysis Pulse-chase experiments were carried out as previously described ( 24 ).

Techniques: Generated, In Vitro, Immunoprecipitation, SDS Page, Western Blot, Flow Cytometry, Fluorescence, Isolation, Cell Culture, Purification, Filtration, Chromatography, Binding Assay, Metabolic Labelling, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Developmental Regulation of Synthesis and Dimerization of the Amyloidogenic Protease Inhibitor Cystatin C in the Hematopoietic System *

doi: 10.1074/jbc.M113.538041

Figure Lengend Snippet: Intracellular ROS from mitochondria promote cystatin C dimerization. A, DC generated in vitro from BM precursors (BM-DC) were incubated with or without the oxidant H2O2 (0.8 mm) for 30 min. ROS levels in the cells were measured by flow cytometry (upper panel) and cystatin C monomers (papain-binding) and dimers (non-binding) were retrieved from cell lysates as described in the legend to Fig. 2A. The data in the histogram shows the mean ± S.E. (n = 3). B, total cell lysates of the H2O2-treated samples were analyzed by non-reducing SDS-PAGE and Western blot. C, BM-DC were incubated at 37 °C with or without the antioxidant-depleting agent EA at 60 μm. Intracellular ROS levels were measured by flow cytometry (upper panel) and cystatin C monomers and dimers were retrieved and visualized by Western blot (lower panel) as in A. D, BM-DC were pulsed chased as described in the legend to Fig. 3A but in the presence or absence of 60 mm EA. Cystatin C monomers and dimers were retrieved from cell lysates after the pulse, and from the culture supernatant after the chase (S/N) as indicated. Newly synthesized cystatin C was visualized by autoradiography. The result is representative of two independent experiments. The intensity of the bands containing cystatin C in the two independent experiments were quantitated by densitometry and shown as mean ± S.E. (bottom graphs). E, BM-DC were either left untreated or treated with 60 μm EA, or 60 μm EA plus 50 nm mitochondria respiration chain complex inhibitor AA in triplicates before they were harvested and loaded with redox sensitive dye to check for intracellular ROS levels by flow cytometry. The data shows the mean ± S.E., which is representative of two independent experiments. F, BM-DC were lysed and their intracellular cystatin C species examined by Western blot as in C. BM-DC from cystatin C-deficient mice (CstC−/−) were included as negative control. All results are representative of two (A, B, D, and E), three (F), or five (C) independent experiments performed.

Article Snippet: Confirmation of transplantation was performed by FACS analysis of splenocytes or blood cells measuring the percentage of donor cells. . ELISA The concentration of cystatin C in serum was determined using the mouse DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. . Radioactive Pulse-Chase Analysis Pulse-chase experiments were carried out as previously described ( 24 ).

Techniques: Generated, In Vitro, Incubation, Flow Cytometry, Binding Assay, SDS Page, Western Blot, Synthesized, Autoradiography, Negative Control

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: (A) The remaining cell viabilities of PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells 48 h after serum withdrawal. When the data were plotted as the percentage of the initial cell viability without serum withdrawal, it was shown that PAC1-CHO had remaining cell viability (57.34±5.91%) that was significantly higher than that of M-PAC1-CHO (36.96±6.85%) or pcDNA-CHO (37.89±7.11%) (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). (B) The intracellular caspase3 activities after serum withdrawal. The reactions of pcDNA-CHO were considered not result from PAC1 because pcDNA-CHO did not express PAC1 or PACAP; therefore, all the data were plotted as fold changes in pcDNA-CHO. As shown, PAC1-CHO had significantly lower caspase3 activity than M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO), whereas there was no significant difference between M-PAC1-CHO and pcDNA-CHO. (C) The intracellular Bcl-2 levels after serum withdrawal. After the data were plotted as the fold changes of pcDNA-CHO, it was shown that PAC1-CHO had significantly higher Bcl-2 level about 2 folds of that in M-PAC1-CHO or pcDNA-CHO (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). The data were represented as the means ± S.E. of three independent experiments. (D) The detection of β-catenin, cyclin D1 and c-myc levels in PAC1-CHO, M-PAC1-CHO and pcDNA-CHO cells by western blotting. The western blotting results and the statistical analysis showed that the levels of β-catenin, cyclin D1 and c-myc (tow targets of β-catenin) in PAC1-CHO cells were significantly higher than those in M-PAC1-CHO or pcDNA-CHO cells (*, P<0.01, PAC1-CHO vs. pcDNA-CHO and M-PAC1-CHO). These findings indicated that overexpression of wild type PAC1 endowed CHO with anti-apoptotic activities against serum withdrawal, suggesting that PAC1 had ligand independent basal activity, while M-PAC1 did not. And Wnt/β-catenin signals were involved in the anti-apoptotic activity of 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: Activity Assay, Western Blot, Over Expression

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) Knockdown of endogenous PACAP and PAC1 with shRNA in Neuro2a. Western blotting assays showed that shRNA against PACAP significantly diminished the expression of endogenous PACAP in neuro2a/PACAP - , and further transfection with shRNA plasmids against PAC1 (+) to neuro2a/PACAP - cells decreased the PAC1 levels significantly, while control plasmids (-) did not interfere with expression of PAC1. The knockdown of PACAP and PAC1 in neuro2a produced a chance for the detection of the correlation of PAC1 down-regulation with its ligand independent basal activity. (B) The remaining cell viabilities of nero2a/PACAP - transfected with PAC1 shRNA plasmids (+) or control plasmid (-). After the data were plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that down-regulation of PAC1 with PAC1 shRNA plasmids (+) decreased the remaining cell viabilities to almost a half of the remaining cell viabilities transfected with control plasmids (-) 48 h after serum withdrawal (*, P<0.01, shRNA + vs. shRNA-). (C) Western blotting of β-catenin, cyclin D1 and c-myc in the nero2a/PACAP - cells transfected with PAC1 shRNA plasmids (+) or control plasmids (-). After the relative protein levels were normalized by the corresponding levels of the control nucleoporin-p62 and plotted as the fold changes in the cells transfected with control plasmids (-), it was shown that PAC1 shRNA plasmids (+) significantly decreased the levels of β-catenin, cyclin D1 and c-myc compared with control plasmids (+)(*, P<0.01, shRNA+ vs. shRNA-). These data suggested that down-regulation of PAC1 in the natural cells such neuro2a with high expression of PAC1 inhibited the anti-apoptotic activities in the ligand free condition. 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: Knockdown, shRNA, Western Blot, Expressing, Transfection, Control, Produced, Activity Assay, Plasmid Preparation

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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

doi: 10.1073/pnas.1801772115

Figure Lengend Snippet: Identification and validation of N6FFA as a modulator of N17 phosphorylation. Cells were methanol fixed and stained with a primary conjugate antibody recognizing N17 with phospho-groups at S13 and S16 (N17-S13pS16p). Five images per well were taken with a 40× air objective. Images were analyzed by PhenoRipper open source software. A unitless PCA plot and a summary of hits are presented in A and B, respectively. Compound 134, indicated by a red arrow, represents vehicle control; compound 50, encircled in green, represents N6FFA. (C) Chemical structure of N6FFA. (D and E) STHdhQ7/Q7 (D) and STHdhQ111/Q111 (E) cells were treated with the indicated concentrations of N6FFA for 24 h, and total cell lysates were separated by SDS/PAGE followed by immunoblotting with N17-S13pS16p antibody. Pixel intensity results from three independent replicates were quantified. Asterisks indicate concentrations of N6FFA that significantly increased N17 phosphorylation compared with control as determined by an unpaired two-tailed t test (*P < 0.05). Bars represent mean values ± SEM. (F) Mouse cortical neurons were transfected with N586-HttQ22 or N586-HttQ82 and were treated with various concentrations of N6FFA. A nuclear condensation assay was performed to determine percent of cell death. Asterisks indicate concentrations of N6FFA that significantly decreased cell death as determined by an unpaired two-tailed t test from three independent replicates (*P < 0.05; **P < 0.001).

Article Snippet: Additional antibodies used in this study were anti–N17-S13pS16p (New England Peptides), anti-CK2 alpha (Abcam), and anti-N6FFA/kinetin riboside (Agrisera) for immunofluorescence and anti-thiophosphate ester (Abcam), anti-GAPDH (Abcam), rabbit anti-huntingtin (PW0595; Enzo Life Sciences), and mouse anti-polyglutamine (mAB1574, Chand mouse anti-huntingtin) for Western blots.

Techniques: Staining, Software, SDS Page, Western Blot, Two Tailed Test, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

doi: 10.1073/pnas.1801772115

Figure Lengend Snippet: CK2 uses KTP as a phospho-donor to phosphorylate huntingtin N17 when primed by phosphorylation. (A and B) Representative blot (A) and quantification (B) of an in vitro N17 phosphorylation assay using CK2 with either ATP or KTP as phospho-donor. **P < 0.001, Mann–Whitney test; n = 3 independent replicates. (C) Representative blot (Upper) and quantification (Lower) of N17-S13pS16p levels in STHdhQ111/Q111 cells upon treatment with N6FFA with or without the addition of the CK2 inhibitor DMAT (4.5 µM). *P < 0.05, Mann–Whitney test (n = 4 independent replicates). (D) Representative blot (Upper) and quantification (Lower) of N17-S13pS16p levels in STHdhQ111/Q111 cells upon treatment with the nonhydrolysable N6FFA derivative 9DK (analysis by Mann–Whitney test) (Left) or treatment with N6FFA (analysis by unpaired t test) (Right). n = 3 independent replicates for both experiments; *P < 0.05. For all data, error bars show mean ± SEM.

Article Snippet: Additional antibodies used in this study were anti–N17-S13pS16p (New England Peptides), anti-CK2 alpha (Abcam), and anti-N6FFA/kinetin riboside (Agrisera) for immunofluorescence and anti-thiophosphate ester (Abcam), anti-GAPDH (Abcam), rabbit anti-huntingtin (PW0595; Enzo Life Sciences), and mouse anti-polyglutamine (mAB1574, Chand mouse anti-huntingtin) for Western blots.

Techniques: In Vitro, Phosphorylation Assay, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

doi: 10.1073/pnas.1801772115

Figure Lengend Snippet: Components of the N6FFA–APRT–KTP–CK2–huntingtin pathway colocalize at sites of DNA damage. (A, Upper) Human fibroblasts were stained with anti-CK2 (green) and anti–N17-S13pS16p (magenta) and were imaged using SR-SIM. (Lower) Magnified views of the boxed areas. (Scale bar: 1 μm.) (B) Human fibroblasts were irradiated with a 405-nm laser in the indicated region to induce DNA damage. After a 20-min incubation period, immunofluorescence was performed against CK2 (green) and N17-S13pS16p (magenta), and the x and y coordinates were revisited to image the irradiated cells by Z-stacked wide-field microscopy and deconvolution. We observed a 100% correlation between CK2 and N17-S13pS16p localization to irradiated regions in two experiments (n = 20–30 cells per experiment). (C) Human fibroblasts were irradiated as in B, stained with anti-N6FFA riboside (green), and imaged by Z-stacked wide-field microscopy and deconvolution. (D) Human fibroblasts were treated with vehicle control (mock) or 10 μM KU55933 for 25 min and then were irradiated as in B, stained with anti-APRT (green) and anti–N17-S13pS16p (magenta), and imaged by Z-stacked wide-field microscopy and deconvolution. (Scale bars: 10 μm in B–D.)

Article Snippet: Additional antibodies used in this study were anti–N17-S13pS16p (New England Peptides), anti-CK2 alpha (Abcam), and anti-N6FFA/kinetin riboside (Agrisera) for immunofluorescence and anti-thiophosphate ester (Abcam), anti-GAPDH (Abcam), rabbit anti-huntingtin (PW0595; Enzo Life Sciences), and mouse anti-polyglutamine (mAB1574, Chand mouse anti-huntingtin) for Western blots.

Techniques: Staining, Irradiation, Incubation, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Urinary α 1 -Antichymotrypsin: A Biomarker of Prion Infection

doi: 10.1371/journal.pone.0003870

Figure Lengend Snippet: Plasma concentration of Cystatin C and α 1 -ACT in patients suffering from AD or sCJD.

Article Snippet: Measurement of human cystatin C was performed using a commercial sandwich ELISA kit (Biovendor Laboratory Medicine Inc., Czech Republic) according to the manufacturer's instructions.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Urinary α 1 -Antichymotrypsin: A Biomarker of Prion Infection

doi: 10.1371/journal.pone.0003870

Figure Lengend Snippet: Cystatin C and α 1 -ACT in patient cerebrospinal fluid.

Article Snippet: Measurement of human cystatin C was performed using a commercial sandwich ELISA kit (Biovendor Laboratory Medicine Inc., Czech Republic) according to the manufacturer's instructions.

Techniques:

Journal: PLoS ONE

Article Title: Urinary α 1 -Antichymotrypsin: A Biomarker of Prion Infection

doi: 10.1371/journal.pone.0003870

Figure Lengend Snippet: Cystatin C, α 1 -ACT and α 1 -microglobulin in the urine of patients.

Article Snippet: Measurement of human cystatin C was performed using a commercial sandwich ELISA kit (Biovendor Laboratory Medicine Inc., Czech Republic) according to the manufacturer's instructions.

Techniques:

Journal: PLoS ONE

Article Title: Urinary α 1 -Antichymotrypsin: A Biomarker of Prion Infection

doi: 10.1371/journal.pone.0003870

Figure Lengend Snippet: Equivalent amounts of CSF protein (10 µg) from late-stage sCJD patients and from appropriate age-matched non-sCJD patients were fractionated under non-reducing SDS-PAGE conditions and analyzed by Western blot using a polyclonal anti-human cystatin C antibody. No difference was found in CSF cystatin C levels of sCJD patients vs. controls.

Article Snippet: Measurement of human cystatin C was performed using a commercial sandwich ELISA kit (Biovendor Laboratory Medicine Inc., Czech Republic) according to the manufacturer's instructions.

Techniques: SDS Page, Western Blot