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: FASEB bioAdvances

Article Title: Calpain-mediated cleavage of p53 in human cytomegalovirus-infected lung fibroblasts.

doi: 10.1096/fba.1028

Figure Lengend Snippet: FIGURE 1 Identification of sub‐53–kDa polypeptides in HCMV‐infected cells, as revealed by a panel of p53 antibodies. Permissive human lung fibroblasts were density arrested and infected with HCMV (5 PFU/cell) or mock infected. Whole‐cell lysates were prepared at the indicated times post infection (PI). Protein aliquots (40 μg/lane) were resolved and membranes probed with the indicated p53 antibodies. Membranes were reprobed with antibody to m‐calpain or actin. These results are representative of at least two independent biological replicates, each one in two technical replicates. Expo: exposure. Arrowhead: p53(ΔCp44). Arrow: p53β. kDa sizes: the relevant molecular weight markers

Article Snippet: The calpain inhibitors E64d (Product Code: IED‐4321‐v) and Z‐Leu‐Leu‐H (ZLLH, Product Code: IZL‐3178‐v) were purchased from Peptides International, Inc (Louisville, KY).

Techniques: Infection, Molecular Weight

Journal: FASEB bioAdvances

Article Title: Calpain-mediated cleavage of p53 in human cytomegalovirus-infected lung fibroblasts.

doi: 10.1096/fba.1028

Figure Lengend Snippet: FIGURE 2 Calpain inhibitors eliminate most p53(ΔCp44) in HCMV‐infected cells. Cells were treated with calpain inhibitors [E64d (100 µM) or ZLLH (100 µM)] at 48 h PI and harvested at the indicated times post treatment (PT). Polypeptides (40 μg/lane) were resolved by SDS‐PAGE. Blots were probed with p53 antibody (DO‐1) and reprobed with actin antibody. These results are representative of at least two independent biological replicates, each one in two technical replicates. Note that the film exposure time for these DO‐1 immunoblots was shorter than those for DO‐1 immunoblots in Figure 1. M, mock‐infected; V, HCMV‐infected. Arrowhead: p53(ΔCp44). kDa sizes: the relevant molecular weight markers

Article Snippet: The calpain inhibitors E64d (Product Code: IED‐4321‐v) and Z‐Leu‐Leu‐H (ZLLH, Product Code: IZL‐3178‐v) were purchased from Peptides International, Inc (Louisville, KY).

Techniques: Infection, SDS Page, Western Blot, Molecular Weight

Journal: FASEB bioAdvances

Article Title: Calpain-mediated cleavage of p53 in human cytomegalovirus-infected lung fibroblasts.

doi: 10.1096/fba.1028

Figure Lengend Snippet: FIGURE 4 Effect of proteasome and/or calpain inhibition on the stability of p53(ΔCp44). Cells were treated with cycloheximide (100 µg/ mL), with or without either the proteasome inhibitor MG132 (10 µM) and/or the calpain inhibitor E64d (100 µM) at 48 hours PI and harvested at the indicated time post treatment (PT). Blots were probed with p53 antibody (DO‐1) and reprobed with m‐calpain antibody. These results are representative of at least two independent biological replicates, each one in two technical replicates. Arrowhead: p53(ΔCp44). kDa sizes: the relevant molecular weight markers

Article Snippet: The calpain inhibitors E64d (Product Code: IED‐4321‐v) and Z‐Leu‐Leu‐H (ZLLH, Product Code: IZL‐3178‐v) were purchased from Peptides International, Inc (Louisville, KY).

Techniques: Inhibition, Molecular Weight

Journal: FASEB bioAdvances

Article Title: Calpain-mediated cleavage of p53 in human cytomegalovirus-infected lung fibroblasts.

doi: 10.1096/fba.1028

Figure Lengend Snippet: FIGURE 5 Sensitivity of p53 in HCMV‐infected cell lysates to cell‐free cleavage by purified µ‐ or m‐calpain and generation of p53(ΔCp44) by purified m‐calpain. HCMV‐infected cells were treated with or without 100 µM E64d 6 hours before harvest to help preserve calpain‐sensitive p53. Nuclear protein (160 µg) was isolated at the indicated time post infection (PI) and incubated with 0.08 units or less of purified µ‐calpain or m‐calpain for 10 minutes at 30°C. In one of the two control samples in each cleavage assay, calpain was replaced with EDTA, which inactivates endogenous Ca2+‐dependent proteases (lane 1). Protein extracts (80 µg) were then analyzed by immunoblot with DO‐1 and reprobed with actin antibody. The results are representative of at least two independent biological replicates, each one in two technical replicates. Arrowhead: p53(ΔCp44). kDa sizes: the relevant molecular weight markers

Article Snippet: The calpain inhibitors E64d (Product Code: IED‐4321‐v) and Z‐Leu‐Leu‐H (ZLLH, Product Code: IZL‐3178‐v) were purchased from Peptides International, Inc (Louisville, KY).

Techniques: Infection, Purification, Isolation, Incubation, Control, Cleavage Assay, Western Blot, Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1α via the N-terminal Pathway

doi: 10.1074/jbc.m110.131615

Figure Lengend Snippet: FIGURE 1. Role of degradation (half-life) and localization of endogenous and exogenous PGC-1. A, HL1 cells were treated with CHX or CHX plus MG132. Cells were lysed at 0, 0.5, 1, 2, and 3 h and were evaluated via SDS-PAGE and Western blotting for PGC-1. The pixels for each band were measured and normalized so that the number of pixels at t 0 was 100%. The log10 of the percent of pixels was plotted versus time for each time point, and the t1⁄2 was calculated from the log of 50%. The t1⁄2 for PGC-1 was 0.3 h and 2 h when MG132 was added. B, 18 h after transfection with full-length PGC-1, HL1 cells were treated with CHX MG132, and degradation was assessed as in A. Expression of exogenous PGC-1 was 200-fold that of endogenous PGC- 1. The t1⁄2 for exogenous PGC-1 was 0.4 h. C, HL1 cells were fixed, and localization of endogenous PGC-1 was visualized by immunofluorescence.

Article Snippet: MG132 (20 M; Peptides International) was added to cells 2 h prior to fixation where indicated.MG132 was prepared as a 10 mM stock solution in dimethyl sulfoxide.

Techniques: SDS Page, Western Blot, Transfection, Expressing, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1α via the N-terminal Pathway

doi: 10.1074/jbc.m110.131615

Figure Lengend Snippet: FIGURE2.RatesofdegradationofPGC182inHL1,HeLa,andC2C12myoblastsandmyotubes.18haftertransfectionwithPGC182HL1(A),HeLa(B),C2C12 myoblasts (C), or C2C12 myotubes (D) were treated with CHX MG132 and half-lives determined as described in Fig. 1 legend. The t1⁄2 for PGC182 was 0.7 h (HL1), 1.0 h (HeLa), 0.5 h (C2C12 myoblasts), and 0.6 h (C2C12 myotubes).

Article Snippet: MG132 (20 M; Peptides International) was added to cells 2 h prior to fixation where indicated.MG132 was prepared as a 10 mM stock solution in dimethyl sulfoxide.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1α via the N-terminal Pathway

doi: 10.1074/jbc.m110.131615

Figure Lengend Snippet: FIGURE 3. Immunofluorescent localization of PGC-1, PGC182, and lysine-less PGC182. 18 h after transfection with PGC-1, PGC182, or lysine- less PGC182, HeLa cells were incubated for 2 h with or without MG132. Cells werethereafterfixed,andlocalizationofPGCwasvisualizedwithanti-PGC-1 via immunofluorescence.

Article Snippet: MG132 (20 M; Peptides International) was added to cells 2 h prior to fixation where indicated.MG132 was prepared as a 10 mM stock solution in dimethyl sulfoxide.

Techniques: Transfection, Incubation, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1α via the N-terminal Pathway

doi: 10.1074/jbc.m110.131615

Figure Lengend Snippet: FIGURE 4. Rates of degradation of PGC182 and its lysine mutants. 18 h after transfection with PGC182 (A), PGC182 K143R/K144R (B), PGC182 K77R/K143R/K144R (C), or PGC182 K54R/K77R/K143R/K144R (lysine-less PGC182) (D), HL1 cells were treated with CHX MG132 and half-lives determined as described in Fig. 1 legend. The t1⁄2 for each of the four PGC182 species was 0.5 h and extended to 3.8–5.5 h with MG132.

Article Snippet: MG132 (20 M; Peptides International) was added to cells 2 h prior to fixation where indicated.MG132 was prepared as a 10 mM stock solution in dimethyl sulfoxide.

Techniques: Transfection

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1α via the N-terminal Pathway

doi: 10.1074/jbc.m110.131615

Figure Lengend Snippet: FIGURE 5. Ubiquitin conjugates accumulate in the presence of MG132. HeLa cells were transfected with PGC182, lysine-less PGC182, or 6-X-LL-myc lysine-less PGC182 together with FLAG-ubiquitin. 18 h later incubation was continued for 2 h MG132. Thereafter, cells were lysed and lysates immuno- precipitated (IP) with anti-PGC-1, separated on SDS-PAGE, and blotted (WB) with anti-FLAG (A). High molecular weight ubiquitin conjugates noted by bracket were quantitated (B).

Article Snippet: MG132 (20 M; Peptides International) was added to cells 2 h prior to fixation where indicated.MG132 was prepared as a 10 mM stock solution in dimethyl sulfoxide.

Techniques: Ubiquitin Proteomics, Transfection, Incubation, SDS Page, High Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Ubiquitin Proteasome-dependent Degradation of the Transcriptional Coactivator PGC-1α via the N-terminal Pathway

doi: 10.1074/jbc.m110.131615

Figure Lengend Snippet: FIGURE 6. Ubiquitin conjugate formation with PGC182 and lysine-less PGC182. HeLa cells were transfected with PGC182, lysine-less PGC182, or 6-X-LL-myc lysine-less PGC182 together with FLAG-ubiquitin. 18 h later, incu- bation was continued for 2 h MG132. Thereafter, either under nonreducing or reducing (350 mM -mercaptoethanol) conditions cells were lysed and lysates immunoprecipitated (IP) with anti-PGC-1 separated on SDS-PAGE and blotted (WB) with anti-FLAG (A), or immunoprecipitated with anti-FLAG andblottedwithanti-PGC-1(B).Lysateswerealsoimmunoprecipitatedwith anti-PGC-1 and blotted with anti-PGC-1 (C) to identify PGC182 and its lysine-less mutants. PGC182, lysine-less PGC182, and 6-X-LL-myc lysine-less PGC182 are noted by arrows. High molecular weight ubiquitin conjugates are noted by brackets.

Article Snippet: MG132 (20 M; Peptides International) was added to cells 2 h prior to fixation where indicated.MG132 was prepared as a 10 mM stock solution in dimethyl sulfoxide.

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, SDS Page, High Molecular Weight

Journal:

Article Title: A Filamentous Phage Associated with Recent Pandemic Vibrio parahaemolyticus O3:K6 Strains

doi:

Figure Lengend Snippet: SDS-PAGE of f237 phage. Lanes: 1, polypeptide SDS-PAGE molecular size standards (in kilodaltons) (Bio-Rad Laboratories, Hercules, Calif.); 2, purified f237 phage. The arrow indicates a major protein band.

Article Snippet: Lanes: 1, polypeptide SDS-PAGE molecular size standards (in kilodaltons) (Bio-Rad Laboratories, Hercules, Calif.); 2, purified f237 phage.

Techniques: SDS Page, Purification

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure1. ThelevelofAoligomersinthebrainofAPOE4/4ADpatientswassignificantlyhighercomparedwithAPOE3/3ADpatients.A,Immunoblottingof50gofTBS-solublefractions from4control,5APOE3/3AD,and5APOE4/4ADprefrontalbrains.Ananti-AmAb82E1revealedAmonomers(arrow)anddimers(arrowhead).B,QuantificationofTBS-solubleAfrom 8control(whitesquares),6APOE2/xAD(darksquares),10APOE3/3AD(darktriangles),and10APOE4/4ADbrains(blackcircles).ThelevelofAinAPOE4/4ADbrainswassignificantly highercomparedwithcontrolbrains,APOE2/xADbrains,andAPOE3/3ADbrains.*p0.05,**p0.01,one-wayANOVAtest(Tukey’sposthoctest).C,Amyloidburden(%)intheprefrontal cortex of 8 control (white squares), 6APOE2/x AD (dark squares), 10APOE3/3 AD (dark triangles), and 10APOE 4/4 AD brains (black circles) analyzed in this study. There is no significant difference amongAPOE2/x AD, betweenAPOE3/3 AD andAPOE4/4 AD brains, one-way ANOVA test (Kruskal–Wallis test).D, Correlation analysis between the level TBS-soluble A and the level of A amyloid burden in 6 APOE 2/x AD (dark squares), 10 APOE 3/3 AD (dark triangles), and 10 APOE 4/4 AD brains (black circles). There is no significant difference. E, QuantificationofapoEconcentrationintheTBS-solublefractionof8control(whitesquares),6APOE2/xAD(darksquares),10APOE3/3AD(darktriangles),and10APOE4/4ADbrains(black circles).F,ImmunoblottingofSEC-separatedfractionsfromAPOE4/4ADbrain.Anti-AmAb82E1and6E10revealedA(arrow)andsAPP(arrowhead).Aelutedfrom94kDato217kDaas HMWAandelutedfrom8.6to16kDaasLMWA.Estimatedmolecularweight(kDa)wasindicatedabove(arrowheads).G,Representativedataoftheseparationof200mgofTBS-solublefractions of APOE 3/3 AD (triangles) and APOE 4/4 AD (squares) brains by double Superdex 75 SEC columns. The concentration of A40 is measured by A specific ELISA (BNT77-BA27) (Wako). Estimated molecular weight (kDa) was indicated above (arrowheads). A in TBS-soluble fraction formed dimer, trimer and HMW oligomers.

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Molecular Weight

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure3. PurifiedapoE-containingHDLparticlesenhancedoligomerformationofsyntheticA1–42invitro.A,Immunoblot- tingforAafterincubationof0.1mg/mlsyntheticA1–42withPBS,10gofpurifiedapoE2,10gofapoE3,or10gofapoE4 fortheindicatedtimes(hours).Anti-AmAb6E10revealedAmonomer,dimer,trimer,andtetramer(arrows).B,Bandintensity of remaining A in SDS-polyacrylamide gels after incubation of synthetic A1–42 oligomers with PBS (no), 5 g/ml purified lipidated apoE2, apoE3, or apoE4 for 12 h using an anti-A mAb 6E10. Lipidated apoE4 significantly increased the level of A trimer and tetramer compared with no lipidated apoE samples. N 6, average SD, *p 0.05, one-way ANOVA test (Bonfer- roni’stest).C,Luminescencefromconditionedmediacontainingsplit-luciferase-taggedAoligomersincubatedwith0,0.1,0.3, 0.6, 1.25, 2.5, 5, or 10 g of purified apoE2 (lipid apoE2, squares), purified apoE3 (lipid apoE3, triangles), or purified apoE4 (lipid apoE4, circles) for 24 h. N 6, average SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). D, Incubation of LMW A isolated from TBS-soluble fractions of the AD brains with (apoE) or without (PBS) 5 g of purified lipid apoE3 and separated the samplesbydoubleSuperdex75SECcolumns.TheconcentrationofA40wasmeasuredbyA-specificELISA(BNT77-BA27,WAKO Chemicals) and obtained the ratio of HMW A measured (in fraction 7 and 8). N 4, average SD, *p 0.05, student’ t test.

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Western Blot, Incubation, Purification, Luciferase, Isolation

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure 4. ApoE enhanced the level of A oligomers in an isoform-dependent manner. A, TransienttransfectionofGFP(control),apoA-II,apoE2,apoE3,orapoE4intodouble-expressing HEK293cells.Luminescenceofconditionedmediawasmeasured.apoE3significantlyincreased the luminescence compared with apoE2 and apoE4 significantly increased the luminescence compared with apoE3. N 6, average SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). B, Immunoblotting of conditioned media from GFP (control), apoE2, apoE3, or apoE4 transiently transfected double-expressing HEK293 cells by an anti-apoE mAb 3H1. C, Transient transfectionofapoA-II,apoE2,apoE3,orapoE4intodouble-expressingHEK293cells.Lumines- cenceofcelllysateswasmeasured.Thereisnosignificantdifferenceoftheluminescenceamong apoE2-, apoE3-, or apoE4-expressing cells. N 6, average SD, one-way ANOVA test (Bon- ferroni’s test). D, Transient transfection of apoA-II, apoE3, apoE4, or apoE4 R61T mutant into double-expressing HEK293 cells. Luminescence of conditioned media was measured. apoE4 significantly increased the luminescence compared with apoE4 R61T mutant. N 6, aver- age SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). E, Transient transfection of apoE2, apoE3, or apoE4 into double-expressing HEK293 cells and separation of conditioned media by a SEC column Superdex 200. Representative data of the luminescence profile of the elutants from conditioned media of apoE2 (circles)-, apoE3 (triangles)-, or apoE4 (squares)- transfected cells. Two peaks, HMW oligomers and dimers (arrows) were observed. F, Average ratiobetweenHMWoligomersanddimers.N 3,averageSD,*p0.01,one-wayANOVA test (Bonferroni’s test).

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Control, Expressing, Western Blot, Transfection, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Apolipoprotein E, Especially Apolipoprotein E4, Increases the Oligomerization of Amyloid Peptide

doi: 10.1523/jneurosci.1542-12.2012

Figure Lengend Snippet: Figure 6. Lipid-binding domain of apoE was necessary for the enhancement of A oligomers. A, Schematic structure of apoE and apoE fragments. The epitopes of mAb 6C5 and mAb 3H1 is illustrated. B, Transient transfection of GFP, apoE3, apoE2 NTF, apoE3 NTF, apoE4 NTF, apoE CTF, or both apoE3 and apoE3 NTF into double-expressing HEK293 cells. Lumines- cence of conditioned media was measured. apoE3 and apoE CTF significantly increased the luminescence, on the other hand, apoE2 NTF, apoE3 NTF, or apoE4 NTF did not increase the luminescence. N 6, average SD, *p 0.05, one-way ANOVA test (Bonferroni’s test). C, Immunoblotting of conditioned media by the anti-apoE mAb 6C5 (top) and 3H1 (bottom panel). MAb 6C5 revealed 36 kDa band (full-length apoE, arrowhead) and 26 kDa band (apoE NTF, arrow). MAb 3H1 revealed also 36 kDa band (full-length apoE, arrowhead) and 10 kDa doublet band (apoE CTF, arrow). D, Transient transfection of GFP, apoA-II, apoE3, apoE3 NTF, apoE CTF, apoE 231–299, apoE 243–299, apoE 192–272, and apoE 192–242 into double-expressing HEK293 cells. Luminescence of conditioned media was measured. apoE3 significantly increasedtheluminescencecomparedwithapoA-II,apoECTFfragments.apoECTFsignificantlyincreasedtheluminescence compared with apoE 192–272 and apoE 192–272 significantly increased the luminescence compared with apoE 192–242. N 6,averageSD,*p0.05,one-wayANOVAtest(Bonferroni’stest).E,TransienttransfectionofGFP,apoA-II,apoE3, apoE3 NTF, apoE CTF, apoE3 243–272, apoE3 273–299, and apoE3 243–299 into double-expressing HEK293 cells. Luminescence of conditioned media was measured. apoE3 and apoE CTF significantly increased the luminescence com- pared with apoA-II. ApoE3 also significantly increased the luminescence compared with three apoE3 deletion mutants. ApoE3 273–299 significantly increased the luminescence compared with apoE3 243–272 or apoE3 243–299. N 6, average SD, *p 0.01, **p 0.05, one-way ANOVA test (Bonferroni’s test).

Article Snippet: We incubated 0.1 mg/ml synthetic A 1– 42 (Peptides International) with or without 10 g of purified apoE2, apoE3, or apoE4 particles at 4°C for 0, 1.5, 6, 12, and 24 h and immediately applied the solution to SDS-PAGE (Hori et al., 2007).

Techniques: Binding Assay, Transfection, Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: Developmental Pathogenicity of 4-Repeat Human Tau Is Lost with the P301L Mutation in Genetically Matched Tau-Transgenic Mice

doi: 10.1523/JNEUROSCI.1256-19.2019

Figure Lengend Snippet: Higher steady-state levels of 4R NM than P301L hTau. A, Relative qRT-PCR shows similar hTau mRNA expression in 8-week-old rT1 and rT2 forebrains. hTau mRNA was not detected in T1 or T2 responders, demonstrating a lack of leaky transgene expression in the absence of the activator transgene (data not shown). B, Densitometric analysis of Western blots shows higher steady-state levels of NM than P301L hTau in forebrains of 8-week-old mice. C, Representative immunoblots for hTau (Tau13 antibody) and GAPDH protein for data in B. Proteins were extracted in RIPA buffer and run on a 10%–20% Tris-HCl gel. D, Soluble tau extracted in TBS buffer and measured by human tau ELISA also shows higher steady-state levels of NM than P301L hTau in 8-week-old forebrains. E, Quantification of full-length (FL) human and mouse tau in phosphatase-treated samples from 8-week-old forebrains, showing higher overexpression of hTau in rT1 (12.6-fold) than rT2 (6.2-fold) relative to tTA+/− mice. F, Representative immunoblots for human and mouse tau (Tau46) and β-III tubulin for data in E. Proteins were extracted in RIPA buffer, treated with phosphatase, and run on a 10.5%–14% Tris-HCl gel. n values are in parentheses. Graphs represent group mean ± SD. ***p < 0.001.

Article Snippet: Protein was immunoblotted with Tau13 (1:60,000, BioLegend), GAPDH (14C10, Cell Signaling Technology, 1:4000), Tau46 (4019T, Cell Signaling Technology, 1:10,000), β-III tubulin (79-720, ProSci, 1:10,000), synaptophysin (SY38 ab8049, Abcam, 1:10,000), PSD-95 (2507S, Cell Signaling Technology, 1:10,000), CP13 (pSer202, Peter Davies, 1:1000), PHF1 (pSer396/404, Peter Davies, 1:1500), rabbit anti-human tau (ab74391, Abcam, 1:10,000), α-tubulin (Thermo Fisher Scientific, DM1A 62204, 1:10,000), MAPK (05-481, Millipore, 1:250), and JNK/SAPK1 (06-748, Millipore, 1:5000) antibodies.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: The Journal of Neuroscience

Article Title: Developmental Pathogenicity of 4-Repeat Human Tau Is Lost with the P301L Mutation in Genetically Matched Tau-Transgenic Mice

doi: 10.1523/JNEUROSCI.1256-19.2019

Figure Lengend Snippet: 4R NM hTau binds MTs more strongly than P301L hTau. A, Validation of protocol to isolate MTs and MAPs in the pellet (P), leaving cytosolic elements in the supernatant (S) with molecular weight in kDa indicated on the left. The cytosolic fractions (B) and MT/MAP fractions (C) were run through SDS-PAGE and immunolabeled with hTau, GAPDH, and β-III tubulin antibodies. Right, Representative images with samples from both female (F) and male (M) mice. Proteins were run on 10.5%–14% gels. Fluorescent signal from Western blots was quantified by densitometry, and values represent averages of two separate experiments (left). Graphs represent group mean ± SD. n values are in parentheses. ***p < 0.001.

Article Snippet: Protein was immunoblotted with Tau13 (1:60,000, BioLegend), GAPDH (14C10, Cell Signaling Technology, 1:4000), Tau46 (4019T, Cell Signaling Technology, 1:10,000), β-III tubulin (79-720, ProSci, 1:10,000), synaptophysin (SY38 ab8049, Abcam, 1:10,000), PSD-95 (2507S, Cell Signaling Technology, 1:10,000), CP13 (pSer202, Peter Davies, 1:1000), PHF1 (pSer396/404, Peter Davies, 1:1500), rabbit anti-human tau (ab74391, Abcam, 1:10,000), α-tubulin (Thermo Fisher Scientific, DM1A 62204, 1:10,000), MAPK (05-481, Millipore, 1:250), and JNK/SAPK1 (06-748, Millipore, 1:5000) antibodies.

Techniques: Biomarker Discovery, Molecular Weight, SDS Page, Immunolabeling, Western Blot