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Image Search Results
Journal: Microbiology Spectrum
Article Title: CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3
doi: 10.1128/spectrum.02758-23
Figure Lengend Snippet: Necroptosis is induced in PBMCs and spleens of CSFV-infected piglets in vivo . ( A ) PBMCs from CSFV-infected and uninfected piglets at 7 dpi were isolated, and necrotic cell features as well as autophagy-like vesicles were visualized by transmission electron microscopy (N, nucleus; M, mitochondria; Go, golgi; ASS, autophagosome). ( B ) Quantitative analysis of the number of autophagosomes observed in ( A ). Error bars indicate the mean (± SD) of five independent experiments. **, P < 0.01 (one-way ANOVA). ( C ) Quantitative analysis of the proportion of necrotic cells observed in ( A ). Error bars indicate the mean (± SD) of three independent experiments. ***, P < 0.001 (one-way ANOVA). ( D ) Spleens from CSFV-infected and uninfected piglets at 7 d were collected, and necrotic cell features as well as autophagy-like vesicles were visualized by transmission electron microscopy (N, nucleus; red arrow, red blood cell). ( E ) Quantitative analysis of the number of autophagosomes observed in ( D ). Error bars indicate the mean (± SD) of five independent experiments. *, P < 0.05 (one-way ANOVA). ( F ) Quantitative analysis of the proportion of necrotic cells observed in ( D ). Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.05 (one-way ANOVA). ( G ) The protein levels of RIPK1, MLKL, TNF -α, IL-1β, and CSFV N pro in PBMC of CSFV-infected and non-infected piglets were detected by Western blot. ( I and K ) The mRNA expressions of RIPK1 , RIPK3 , MLKL , TNF-α , T NF-R1 , ZBP1, and Caspase-8 in PBMC of CSFV-infected and non-infected piglets were determined by qRT-PCR. The fold changes were related to the internal control β-actin. Error bars indicate the mean (± SD) of three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA). ( J and L ) The mRNA expressions of RIPK1 , RIPK3 , MLKL , TNF-α, and ZBP1 in spleens of CSFV-infected and non-infected piglets were determined by qRT-PCR. The fold changes were related to the internal control β-actin. Error bars indicate the mean (± SD) of three independent experiments. ***, P < 0.001 and ****, P < 0.0001 (two-way ANOVA). ( H ) Flow cytometry was used to determine the proportion of necrotic cells in PBMCs from CSFV-infected and uninfected piglets at 7 d. ( M ) The expression of CSFV E2 in the spleen of infected and uninfected piglets was detected by IHC. ( N ) The expression of RIPK1, RIPK3, and MLKL in the spleen of infected and uninfected piglets was detected by IHC. During this period, we fixed, embedded, and sectioned spleen tissues. The sections were then stained with specific antibodies against RIPK1, RIPK3, and MLKL, respectively. The appearance of similar contours in the sliced images is a result of the proximity of the tissue sections. ( O ) Immunohistochemical scoring of the detection indices in ( N ).
Article Snippet: The primary antibodies used in this study were as follows: mouse monoclonal anti-TNF-α (Santa Cruz Biotechnology, sc-52746), mouse monoclonal anti-IL-1β (Santa Cruz Biotechnology, sc-32294), rabbit monoclonal anti-TRIM25 (Abcam, ab167154),
Techniques: Infection, In Vivo, Isolation, Transmission Assay, Electron Microscopy, Western Blot, Quantitative RT-PCR, Flow Cytometry, Expressing, Staining, Immunohistochemistry
Journal: Microbiology Spectrum
Article Title: CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3
doi: 10.1128/spectrum.02758-23
Figure Lengend Snippet: Necroptosis was induced at the early stage and inhibited at the late stage of CSFV infection in vitro . To establish a PBMC cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( A ) and qRT-PCR ( G ). ( B ) Gray-scale value analysis of the detection indexes in ( A ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a PK-15 cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis marker genes including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( C ) and qRT-PCR ( H ). ( D ) Gray-scale value analysis of the detection indexes in ( C ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA). To establish a 3D4/21 cell model of CSFV infection in vitro , cells were collected at 24, 48, and 60 h post-infection, and protein expression and mRNA levels of necroptosis markers including RIPK1, RIPK3, MLKL, and ZBP1 were detected by Western blot ( E ) and qRT-PCR ( I ). ( F ) Gray-scale value analysis of the detection indexes in ( E ); the relative intensity was related to the internal control Tubulin. Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05*; P < 0.05; **, P < 0.01; and ***, P < 0.001 (two-way ANOVA).
Article Snippet: The primary antibodies used in this study were as follows: mouse monoclonal anti-TNF-α (Santa Cruz Biotechnology, sc-52746), mouse monoclonal anti-IL-1β (Santa Cruz Biotechnology, sc-32294), rabbit monoclonal anti-TRIM25 (Abcam, ab167154),
Techniques: Infection, In Vitro, Expressing, Western Blot, Quantitative RT-PCR, Marker
Journal: Microbiology Spectrum
Article Title: CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3
doi: 10.1128/spectrum.02758-23
Figure Lengend Snippet: CSFV NS4A interacts with RIPK3 and TRIM25 to inhibit necroptosis. ( A ) HEK-293T cells were co-transfected with HA-RIPK3 together with pGFP-C1, GFP-P7, GFP-C, GFP-NS4A, GFP-NS4B, or GFP-NS5A for 24 h, fixed cells were incubated with anti-HA tag primary antibody and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG secondary antibody (red), and nuclei were stained with DAPI. Scale bars: 25 µm. ( B ) pGFP-C1, GFP-P7, GFP-C, GFP-NS4A, GFP-NS4B, and GFP-NS5A were transfected into PK-15 cells for 24 h, and the cells were lysed. The expression levels of viral proteins, RIPK1, RIPK3, and MLKL, were detected by Western blot. The right panel was gray-scale value analysis, the relative intensity was related to the internal control GAPDH. Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA). ( C ) FLAG-RIPK3 was co-transfected with pEGFP-C1 and GFP-NS4A, respectively, into PK-15 cells, and the cells were lysed; Co-IP assay and Western blot analysis were performed. ( D ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in HEK-293T cells, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A, cells were lysed; Co-IP experiments and Western blot analysis were performed. ( E ) PK-15 cells were co-transfected with three constructs, HA-RIPK3, Myc-TRIM25 with GFP-NS4A, and a control construct of cells co-transfected with HA-RIPK3, Myc-TRIM25 with pEGFP-C1, HA-RIPK3, pCMV-N1-Myc with GFP-NS4A, Myc-TRIM25, pCAGGs-HA with GFP-NS4A, The fixed cells were incubated with anti-HA primary antibody and anti-Myc primary antibody, respectively, and then stained with Alexa fluor 555-conjugated anti-Rabbit-IgG Mouse antibody (red) and Dylight 405-labeled anti-Rabbit-IgG secondary antibody (blue) (left panel). Scale bars: 10 µm. The fluorescence intensity profiles of HA-RIPK3 (blue), GFP-NS4A(green), and Myc-TRIM25(red) were analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). ( F ) Myc-TRIM25 was co-transfected with pEGFP-C1 and GFP-NS4A in PK-15 cells, while a control group of cells was set up in which pCMV-N1-Myc was co-transfected with pEGFP-C1 and GFP-NS4A; the cell lysates were detected by Western blot. The gray-scale value analysis was related to the internal control GAPDH (lower panel). Error bars indicate the mean (± SD) of three independent experiments. ****, P < 0.0001 (two-way ANOVA).
Article Snippet: The primary antibodies used in this study were as follows: mouse monoclonal anti-TNF-α (Santa Cruz Biotechnology, sc-52746), mouse monoclonal anti-IL-1β (Santa Cruz Biotechnology, sc-32294), rabbit monoclonal anti-TRIM25 (Abcam, ab167154),
Techniques: Transfection, Incubation, Staining, Expressing, Western Blot, Co-Immunoprecipitation Assay, Construct, Labeling, Fluorescence
Journal: Microbiology Spectrum
Article Title: CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3
doi: 10.1128/spectrum.02758-23
Figure Lengend Snippet: CSFV inhibits necroptosis through autophagy/mitophagy and necroptosis-restricted CSFV infection in vitro . ( A ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h, rapamycin was treated for 6 h, and an uninfected cell control was set up. Cell lysates were then detected by Western blot (left panel). Relative intensity related to internal control Tubulin was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; *, P < 0. 1; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA). ( B ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h, CCCP treated for 6 h, and an uninfected cell control was set up. Cell lysates were then detected by Western blot (left panel). Relative intensity related to internal control Tubulin was analyzed using Image J plugin and plotted by GraphPad Prism 9 (right panel). Error bars indicate the mean (± SD) of three independent experiments. ns, P ≥ 0.05; *, P < 0. 1; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA). ( C ) PK-15 cells were infected with CSFV (MOI = 1) for 48 h and treated with TSZ (20 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD-Fmk) alone or TSZ together with rapamycin for 6 h, and a control of uninfected cells was set up. Necrotic cells were identified by flow cytometry with PI and Annexin V staining. A representative plot of the data is shown in the left panel, and the mean (± SD) of three independent experiments is shown in the right panel. *, P < 0. 1 and ****, P < 0.0001 (two-way ANOVA). ( D ) PK-15 cells infected with CSFV (MOI = 1) were transfected with Flag-RIPK3 for 24 h and then treated with 3-methyladenine (3-MA, 5 mM), chloroquine (CQ, 20 µM), or bafilomycin A1 (Baf A1, 100 nM) for 6 h, and cell lysates were detected by Western blot (upper panel). Flag-RIPK3 was co-transfected with shNC, shATG5, shBECN1, and shLC3 into CSFV-infected PK-15 cells, and cell lysates were detected by Western blot (lower panel). ( E ) PK-15 (left panel) and 3D4/21 (right panel) cells infected with CSFV (MOI = 1) were transfected with HA-RIPK3 or MLKL for 48 h, and pCAGGs-HA was transfected with a control group. The cell supernatants were collected for TCID50 assay. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1 and **, P < 0.01 (one-way ANOVA). ( F ) PK-15 cells infected with CSFV (MOI = 1) were treated with TSZ for 6 h, and control of uninfected cells was set up. The mRNA expressions of RIPK1 , RIPK3 , MLKL, and CSFV NS5B in the cell lysates were determined by qRT-PCR. The fold changes were related to the internal control β-actin. Error bars indicate the mean (± SD) of three independent experiments. *, P < 0.1; **, P < 0.01; ***, P < 0.001 (one-way ANOVA). ( G ) PK-15 cells infected with CSFV (MOI = 1) were treated with increasing amounts of TSZ (wedge shaped, 0; 20 ng/mL TNF-α, 100 nM Smac mimetic, and 10 µM z-VAD-Fmk; 40 ng/mL TNF-α, 100 nM Smac mimetic, and 10 µM z-VAD-Fmk; 40 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD-Fmk) for 6 h, and the cell lysates were detected by Western blot. ( H ) PK-15 cells were treated with increasing amounts of necrosulfonamide (NSA; wedge shaped, 0, 1, 5, and 10 µM), and the cell lysates were detected by Western blot (upper panel). PK-15 cells infected with CSFV (MOI = 1) were treated with NSA (5 µM), and dimethyl sulfoxide (DMSO) was treated with a control group. The cell lysates were detected by Western blot (lower panel). ( I ) PK-15 (left panel) and 3D4/21 (right panel) cells were infected with CSFV (MOI = 1) for 48 h and treated with TSZ (20 ng/mL TNF-α, 100 nM Smac mimetic, and 20 µM z-VAD -Fmk) alone or TSZ together with rapamycin for 6 h, and a control of uninfected cells was set up. The cell lysates were detected by Western blot.
Article Snippet: The primary antibodies used in this study were as follows: mouse monoclonal anti-TNF-α (Santa Cruz Biotechnology, sc-52746), mouse monoclonal anti-IL-1β (Santa Cruz Biotechnology, sc-32294), rabbit monoclonal anti-TRIM25 (Abcam, ab167154),
Techniques: Infection, In Vitro, Western Blot, Flow Cytometry, Staining, Transfection, TCID50 Assay, Quantitative RT-PCR