anti total vp1 antibody  (Cell Signaling Technology Inc)

Journal: Antiviral research

Article Title: Antiviral activity of the HSP90 inhibitor VER-50589 against enterovirus 71.

doi: 10.1016/j.antiviral.2023.105553

Figure Lengend Snippet: Fig. 1. VER-50589 exhibits antiviral effects against EV71 in vitro. (A) Chemical structure of VER-50589. (B–E) RD and HeLa cells were cultured with five- fold dilutions of VER-50589 or ribavirin. After 48 h, cytotoxicity was assessed using an MTT assay. (F–H) RD cells were infected with EV71 (MOI = 0.1) for 2 h and incubated with five-fold dilutions of VER-50589 or ribavirin for 20 h. VP1 protein levels were measured using western blotting (F). EV71 mRNA levels were evaluated using qPCR (G, H). (l) Immu nofluorescence assay detected EV71 mRNA in EV71- infected RD cells treated with VER-50589. (J) EV71- infected RD cells were treated with different con centrations of VER-50589 for 48 h, and the cell freeze-thaw liquid was harvested for the plaque assay. (K)The number of infectious clones of the samples was calculated. (L–M) EV71-infected RD cells were treated with VER-50589 (4 μM) or ribavirin (4 μM) under the same experimental conditions. VP1 protein and viral mRNA levels were measured using western blotting and qPCR, respectively. ***P < 0.001; **P < 0.01; *P < 0.05.

Article Snippet: The following antibodies were used according to manufacturer’s recommendations: anti-phospho-AKT (CST, Cat: 4060S), anti-total AKT (CST, Cat: 4691S), anti-HSP90 (Abcom, Cat: ab208035), anti-phospho-ERK1/2 (Santa Cruz, Cat: sc-81492), anti-total ERK1/2 (Santa Cruz, Cat: sc-514302), anti-phospho-RAF (CST, Cat: 2969S), antitotal RAF (CST, Cat: 148145S), anti-total VP1 antibody (CST, cat: 9167), goat anti-rabbit IgG (Cat: SA00001-2, Proteintech, China), goat antimouse IgG (Cat: SA00001-1, Proteintech, China), β-actin (Cat: 60008- 1, Proteintech, China), and GAPDH (Cat: 10494-1, Proteintech, China).

Techniques: In Vitro, Cell Culture, MTT Assay, Infection, Incubation, Western Blot, Plaque Assay, Clone Assay

Journal: Antiviral research

Article Title: Antiviral activity of the HSP90 inhibitor VER-50589 against enterovirus 71.

doi: 10.1016/j.antiviral.2023.105553

Figure Lengend Snippet: Fig. 4. VER-50589 inhibits HSP90 and mediates AKT and RAF phosphorylation. (A–B) RD cells were transfected with HSP90-specific siRNA and cultured for 24 h. The p-AKT, AKT, RAF, p-RAF, and HSP90 levels were detected using western blotting (A). HSP90 mRNA levels were detected using qPCR (B). (C–D) RD cells were transfected with HSP90-specific siRNA for 24 h and infected with EV71 (MOI = 8) for 24 h. The VP1, p-AKT, AKT, p-RAF, RAF, and HSP90 levels were detected using western blotting (C). The mRNA levels of EV71 were detected using qPCR (D). (E) Cherry-HSP90 plasmid-carrying RD cells were infected with EV71 (MOI = 8) for 24 h. The expression levels of HSP90, p-AKT, AKT, p-RAF, RAF, and VP1 were detected using western blotting. (F) RD cells were infected with the EV71 virus with different MOI (0.01, 0.1, 1, and 10) and subjected to western blotting. (G) RD cells were infected with EV71 (MOI = 0.1) and harvested at 8 h, 12 h, 18 h, and 22 h. Protein levels were detected using western blotting. (H) RD cells were treated with five-fold dilution of VER-50589 and cultured for 20 h. Different protein levels were detected using western blotting. (I) RD cells were incubated with EV71 (MOI = 0.1) for 2 h, and the medium was replaced with one containing a five-fold series dilution of VER-50589 for another 20 h incubation. Proteins were extracted for western blotting analysis.

Article Snippet: The following antibodies were used according to manufacturer’s recommendations: anti-phospho-AKT (CST, Cat: 4060S), anti-total AKT (CST, Cat: 4691S), anti-HSP90 (Abcom, Cat: ab208035), anti-phospho-ERK1/2 (Santa Cruz, Cat: sc-81492), anti-total ERK1/2 (Santa Cruz, Cat: sc-514302), anti-phospho-RAF (CST, Cat: 2969S), antitotal RAF (CST, Cat: 148145S), anti-total VP1 antibody (CST, cat: 9167), goat anti-rabbit IgG (Cat: SA00001-2, Proteintech, China), goat antimouse IgG (Cat: SA00001-1, Proteintech, China), β-actin (Cat: 60008- 1, Proteintech, China), and GAPDH (Cat: 10494-1, Proteintech, China).

Techniques: Phospho-proteomics, Transfection, Cell Culture, Western Blot, Infection, Plasmid Preparation, Expressing, Virus, Incubation

Journal: Oncoimmunology

Article Title: Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling

doi: 10.1080/2162402X.2017.1321185

Figure Lengend Snippet: Simultaneous downregulation of PTP1B and TC-PTP enhances moDCs maturation. (A) Comparisons between double heterozygous (1B:TC fl/wt ) and TC fl/wt moDCs, 1B fl/wt moDCs and wt moDCs respectively. MFI values of the expression of MHC class I, CD86 and CD80 ( n = 5). (B) Production of Th1 polarizing cytokines ( n = 5): (B) IL-12 and (C) IFNγ. (D) Quantification of IFNγ produced by activated OT-I CD8 + T cells co-cultured with mature and OVA-pulsed 1B:TC fl/wt, 1B fl/wt , TC fl/wt or wt moDCs for 48 h ( n = 8–9). (E) Quantification of IFNγ produced by activated OT-II CD4 + T cells co-cultured with mature and OVA-pulsed 1B:TC fl/wt , 1B fl/wt , TC fl/wt or wt moDCs for 48 h ( n = 5). (F) Activation status of Src kinase and IkBa downstream of TLR4 ( n = 3). Significant differences are represented by p values and (*) indicate significant differences between wt DCs and the rest of the groups. (G) Activation status of STAT1, STAT4 and Src in STAT1 inhibitor (fludarabine) or STAT4 inhibitor (lisofylline) treated or non-treated 1B:TC fl/wt , 1B fl/wt , TC fl/wt or wt moDCs ( n = 3). Production of Th1-polarizing cytokines by 1B:TC fl/wt , 1B fl/wt , TC fl/wt or wt moDCs previously treated with STAT1- or STAT4-inhibitor during the maturation process ( n = 3): (H) IL-12 and (I) IFNγ. The results are representative of at least three independent experiments. Significant differences among the groups are represented by p values and (*) indicate significant differences within a group. The comparisons were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric.

Article Snippet: The following proteins were detected using antibodies from Cell Signaling Technologies (CST) phospho-specific and total STAT1 (total-stat1 CST cat# 9172 and phospho-stat1 CST cat# 9134), STAT3 (total-stat3 CST cat# 9139 and phosphor-stat3 CST cat# 9145), STAT5 (total-stat5 CST cat# 9310 and phosphor-stat5 CST cat# 9356), STAT4 (total-stat4 CST cat# 2653 and phosphor-stat4 CST cat# 5267), Src (total-Src CST cat# 2109 and phospho-Src CST cat# 2101), IkBα (total-IkBα CST #4812 and phospho-IkBα CST #2859), ERK (total-ERK CST cat# 9102 and phospho-ERK CST cat# 9106, p38 (total-p38 CST #9212 and phospho-p38 CST cat# 9211) and c-Jun (total-cJun CST cat# 9165 and phospho-cJun CST cat# 9261) proteins were detected using polyclonal antibodies (Cell Signaling Technology). β-Actin and Calnexin (a gift from Dr. John J.M.

Techniques: Expressing, Produced, Cell Culture, Activation Assay

Journal: Oncoimmunology

Article Title: Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling

doi: 10.1080/2162402X.2017.1321185

Figure Lengend Snippet: Pharmacological inhibition of both PTP1B and TC-PTP with specific inhibitor. (A) Specificity of 1B/TC inh (50 μM) to inhibit PTP1B and TC-PTP dephosphorylation ( n = 3). (B) Titration of 1B/TC inh in mouse moDCs using IL-12 production as indicator of activation ( n = 3). (C) IFNγ production by 1B/TC inh-treated mature moDCs (4 μM dose) ( n = 4). (D) Expression levels (MFI) of CD40, CD80, CD86, MHC class I and II on 1B/TC inh-treated mature moDCs (4 μM dose) ( n = 3). (E) Activation status STAT1 and STAT4 ( n = 3). (F) Activation status of Src kinase and IkBα ( n = 4). Therapeutic properties of 1B/TC inh-treated moDCs in a mouse model of E.G7 lymphoma: (G) Tumor volume before moDC treatment (10 d after implantation of 5 × 10 5 E.G7-OVA cells) and (H) after intraperitoneal (IP) injections of 5 × 10 6 1B/TC inh-treated or non-treated moDCs compared with control group (tumor-bearing mice without moDC treatments). The IP injections with moDCs were given 18 d after tumor implantation. (I) Images represent tumor-bearing mice 8 d after of moDC treatment. The results are representative of at least three independent experiments. The differences in more than two groups were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The differences between two groups were determined with unpaired t test (two tails of distribution). Significant differences are represented by p values <0.05.

Article Snippet: The following proteins were detected using antibodies from Cell Signaling Technologies (CST) phospho-specific and total STAT1 (total-stat1 CST cat# 9172 and phospho-stat1 CST cat# 9134), STAT3 (total-stat3 CST cat# 9139 and phosphor-stat3 CST cat# 9145), STAT5 (total-stat5 CST cat# 9310 and phosphor-stat5 CST cat# 9356), STAT4 (total-stat4 CST cat# 2653 and phosphor-stat4 CST cat# 5267), Src (total-Src CST cat# 2109 and phospho-Src CST cat# 2101), IkBα (total-IkBα CST #4812 and phospho-IkBα CST #2859), ERK (total-ERK CST cat# 9102 and phospho-ERK CST cat# 9106, p38 (total-p38 CST #9212 and phospho-p38 CST cat# 9211) and c-Jun (total-cJun CST cat# 9165 and phospho-cJun CST cat# 9261) proteins were detected using polyclonal antibodies (Cell Signaling Technology). β-Actin and Calnexin (a gift from Dr. John J.M.

Techniques: Inhibition, De-Phosphorylation Assay, Titration, Activation Assay, Expressing, Tumor Implantation

Journal: Oncoimmunology

Article Title: Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling

doi: 10.1080/2162402X.2017.1321185

Figure Lengend Snippet: Application of 1B/TC inh-treated human DCs in cancer immunotherapy. (A) Activation status of STAT1 and STAT4 in 1B/TC inh-treated human moDCs derived from a pancreatic cancer (PaC) patient ( n = 3). (B) IL-12 production by 1B/TC inh-treated human moDCs from four PaC patients. (C) Frequency of antigen-specific (CEA and CA19–9) activated T cells measured based on IFNγ production detected in ELISpot assay. The differences in more than two groups were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The differences between two groups were determined with unpaired t -test (two tails of distribution). Significant differences are represented by p values <0.05.

Article Snippet: The following proteins were detected using antibodies from Cell Signaling Technologies (CST) phospho-specific and total STAT1 (total-stat1 CST cat# 9172 and phospho-stat1 CST cat# 9134), STAT3 (total-stat3 CST cat# 9139 and phosphor-stat3 CST cat# 9145), STAT5 (total-stat5 CST cat# 9310 and phosphor-stat5 CST cat# 9356), STAT4 (total-stat4 CST cat# 2653 and phosphor-stat4 CST cat# 5267), Src (total-Src CST cat# 2109 and phospho-Src CST cat# 2101), IkBα (total-IkBα CST #4812 and phospho-IkBα CST #2859), ERK (total-ERK CST cat# 9102 and phospho-ERK CST cat# 9106, p38 (total-p38 CST #9212 and phospho-p38 CST cat# 9211) and c-Jun (total-cJun CST cat# 9165 and phospho-cJun CST cat# 9261) proteins were detected using polyclonal antibodies (Cell Signaling Technology). β-Actin and Calnexin (a gift from Dr. John J.M.

Techniques: Activation Assay, Derivative Assay, Enzyme-linked Immunospot

Journal: Oncoimmunology

Article Title: Immunosuppressive CD14 + HLA-DR lo/neg monocytes are elevated in pancreatic cancer and “primed” by tumor-derived exosomes

doi: 10.1080/2162402X.2016.1252013

Figure Lengend Snippet: Mechanism of HLA-DR downregulation on monocytes mediated by PC-Exo. (A) CD14+ monocytes were co-incubated with 50 µg of PANC-1 exosomes or exosomes that had been treated with NAC for 4 d. Reactive oxygen species (ROS, shown as green) were detected using the Cell ROX® detection assay. Scale bar, 20 µm. The right panel shows zoomed images (4×). Scale bar, 5 µm. (B) Quantification of ROS (green) in control and treatment groups. (C) mRNA expression of arginase I in CD14+ monocytes after treatment with 50 µg PANC-1 exosomes (+Exo) compared to untreated cells (−Exo). (D) Exosomes isolated from two PC patient plasma samples (PC1 and PC2 Exo) and PANC-1 Exo were co-incubated with monocytes. pSTAT1, pSTAT3, total STAT3, and total STAT1 protein expression levels were assessed compared to untreated monocytes (−Exo). **p < 0.001; ***p < 0.0001.

Article Snippet: Samples were probed with phospho-STAT1, phospho-STAT3, total STAT1, and total STAT3 (Cell Signaling Technology, no. 9167S, 9145P, 9172P, 4904P).

Techniques: Incubation, Detection Assay, Expressing, Isolation

Journal: Molecular cancer therapeutics

Article Title: Enhancing Therapeutic Efficacy of Oncolytic Herpes Simplex Virus-1 with Integrin β1 Blocking Antibody OS2966

doi: 10.1158/1535-7163.MCT-18-0953

Figure Lengend Snippet: A-B) Impact of OS2966 on oHSV-induced anti-viral signaling. A) Real time PCR analysis for changes in gene expression of IFNα, IFNβ, and stat1 gene in both patient derived primary GBM (GBM30) and breast cancer cells 24 hours post oHSV treatment. Data presented are fold changes in gene expression ± SD relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B) Real time PCR analysis for changes in a set of the type-I IFN responsive genes involved in the antiviral defense in both patient derived primary GBM (GBM30) and breast cancer cells 24 hours post oHSV treatment. Data presented are relative gene expression between oHSV+OS2966 and oHSV + control IgG. *, p < 0.05. Comparisons made between control IgG versus OS2966 and oHSV versus oHSV+OS2966 treated cells. C) OS2966 inhibits oHSV-induced CCN1 expression and Stat1 activation. Patient derived primary GBM (GBM30) cells were infected with/without 34.5ENVE (MOI=0.01). Two hours post oHSV infection, unbound viruses were removed and 10 ug/ml of control IgG/OS2966 were added and cultured for 24 hours. Cell lysates were probed with antibodies against CCN1, phosphor-Stat1, and total Stat1. β-tubulin was used as a loading control.

Article Snippet: After blocking, the membrane was incubated with primary antibodies against CCN1 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-Stat1 and total Stat1 (BD Transduction Laboratories, San Diego, CA); β-tubulin (Cell Signaling Technology, Waltham, MA); phospho-FAK (S359), total FAK, ITGB1 and GAPDH (Abcam, Cambridge, MA) (each diluted 1:1000) followed by either HRP-conjugated secondary anti-mouse antibody (each diluted 1:1000) (GE Healthcare, Piscataway, NJ) or HRP-conjugated secondary goat anti-rabbit antibody (each diluted 1:1000) (Dako, Hamburg, Germany).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Activation Assay, Infection, Cell Culture