mouse rip3 (ProSci Incorporated)
ProSci Incorporated is a verified supplier 
ProSci Incorporated manufactures this product 
94
Structured Review
ProSci Incorporated
mouse rip3
Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse rip3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse rip3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse rip3 - by Bioz Stars,
2023-06
94/100 stars
Images
1) Product Images from "Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons"
Article Title: Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons
Journal: BioMed Research International
doi: 10.1155/2014/290182
Figure Legend Snippet: The regulation of RIP3 in TNF- α -induced toxicity of hippocampal neurons in vivo . (a) Nissl staining of hippocampal neurons 72 h after treatment. Wild-type (WT) and RIP3 knockout (KO) mice received intracerebroventricular injection of PBS or the indicated dose of TNF- α . The neurons of brain sections from WT and KO mice were analyzed by Nissl staining ( n = 7) and morphology of hippocampal CA3 region was shown. Arrows indicate the loss of hippocampal neurons. (b) and (c) Expressions of RIP1, RIP3, and caspase-3 in the hippocampus after TNF - α treatment. Proteins extracted from hippocampus in the wild-type mice treated with PBS or TNF- α were analyzed by western blot using indicated antibodies. PC: MEF cells were treated with staurosporine at 150 nM for 15 hours. The results shown here are representative of five mice.
Techniques Used: In Vivo, Staining, Knock-Out, Injection, Western Blot
Figure Legend Snippet: TNF- α -induced necrosis of HT-22 cells depends on RIP3 and its kinase activity. (a) HT-22 cells were transfected with the negative control (NC) or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for another 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. (b) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. (c) HT-22 cells stably expressing a siRNA-resistant WT-RIP3 or RIP3-K51A or RIP3-RHIM-Mut were transfected with the control or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. WT-RIP3: HT-22 cells stably expressing a siRNA-resistant wild-type form of RIP3; RIP3-K51A: HT-22 cells stably expressing a siRNA-resistant RIP3 kinase dead mutant. RIP3-RHIM Mut: HT-22 cells stably expressing a siRNA-resistant RHIM domain mutant form of RIP3. (d) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. All experiments were repeated three times with similar results.
Techniques Used: Activity Assay, Transfection, Negative Control, Standard Deviation, Western Blot, Stable Transfection, Expressing, Mutagenesis
mouse rip3 (ProSci Incorporated)
ProSci Incorporated is a verified supplier
ProSci Incorporated manufactures this product
94
Structured Review
ProSci Incorporated
mouse rip3
Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse rip3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse rip3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse rip3 - by Bioz Stars,
2023-06
94/100 stars
Images
1) Product Images from "Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons"
Article Title: Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons
Journal: BioMed Research International
doi: 10.1155/2014/290182
Figure Legend Snippet: The regulation of RIP3 in TNF- α -induced toxicity of hippocampal neurons in vivo . (a) Nissl staining of hippocampal neurons 72 h after treatment. Wild-type (WT) and RIP3 knockout (KO) mice received intracerebroventricular injection of PBS or the indicated dose of TNF- α . The neurons of brain sections from WT and KO mice were analyzed by Nissl staining ( n = 7) and morphology of hippocampal CA3 region was shown. Arrows indicate the loss of hippocampal neurons. (b) and (c) Expressions of RIP1, RIP3, and caspase-3 in the hippocampus after TNF - α treatment. Proteins extracted from hippocampus in the wild-type mice treated with PBS or TNF- α were analyzed by western blot using indicated antibodies. PC: MEF cells were treated with staurosporine at 150 nM for 15 hours. The results shown here are representative of five mice.
Techniques Used: In Vivo, Staining, Knock-Out, Injection, Western Blot
Figure Legend Snippet: TNF- α -induced necrosis of HT-22 cells depends on RIP3 and its kinase activity. (a) HT-22 cells were transfected with the negative control (NC) or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for another 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. (b) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. (c) HT-22 cells stably expressing a siRNA-resistant WT-RIP3 or RIP3-K51A or RIP3-RHIM-Mut were transfected with the control or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. WT-RIP3: HT-22 cells stably expressing a siRNA-resistant wild-type form of RIP3; RIP3-K51A: HT-22 cells stably expressing a siRNA-resistant RIP3 kinase dead mutant. RIP3-RHIM Mut: HT-22 cells stably expressing a siRNA-resistant RHIM domain mutant form of RIP3. (d) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. All experiments were repeated three times with similar results.
Techniques Used: Activity Assay, Transfection, Negative Control, Standard Deviation, Western Blot, Stable Transfection, Expressing, Mutagenesis
anti mouse ripk3 (ProSci Incorporated)
ProSci Incorporated is a verified supplier
ProSci Incorporated manufactures this product
94
Structured Review
ProSci Incorporated
anti mouse ripk3
Anti Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Anti Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti mouse ripk3 - by Bioz Stars,
2023-06
94/100 stars
Images
anti mouse ripk3 (ProSci Incorporated)
ProSci Incorporated is a verified supplier
ProSci Incorporated manufactures this product
94
Structured Review
ProSci Incorporated
anti mouse ripk3
![TNF treatment induces activation of AMPK. ( A ) L929 cells were transfected with non-targeting (siCtrl) or <t>Ripk3</t> siRNAs (si Ripk3 ). 48 h post transfection, cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for the indicated times. Then, whole cell lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. A compilation of representative immunoblots is shown; three ACTB immunblots are shown, but each protein was normalized to its corresponding loading control. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (scr, 0 h TQ). Results are mean + SD from at least 3 independent experiments. Statistical analysis was done by repeated measures two-way ANOVA (corrected by Sidak’s multiple comparisons test between siRNAs and corrected by Tukey’s multiple comparisons test between time points). Statistically significant differences within non-targeting siRNA-transfected cells (compared to scr, 0 h TQ) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001. ( B ) Ripk3 WT and KO MEFs were exposed to indicated treatments (medium [M], 30 ng/ml TNF [T], 100 nM SMAC-mimetic [S], 20 µM z-VAD [Z]) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6653/pmc08726653/pmc08726653__KAUP_A_1899667_F0001_C.jpg)
Anti Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Anti Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti mouse ripk3 - by Bioz Stars,
2023-06
94/100 stars
Images
1) Product Images from "TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy"
Article Title: TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy
Journal: Autophagy
doi: 10.1080/15548627.2021.1899667
Figure Legend Snippet: TNF treatment induces activation of AMPK. ( A ) L929 cells were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (si Ripk3 ). 48 h post transfection, cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for the indicated times. Then, whole cell lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. A compilation of representative immunoblots is shown; three ACTB immunblots are shown, but each protein was normalized to its corresponding loading control. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (scr, 0 h TQ). Results are mean + SD from at least 3 independent experiments. Statistical analysis was done by repeated measures two-way ANOVA (corrected by Sidak’s multiple comparisons test between siRNAs and corrected by Tukey’s multiple comparisons test between time points). Statistically significant differences within non-targeting siRNA-transfected cells (compared to scr, 0 h TQ) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001. ( B ) Ripk3 WT and KO MEFs were exposed to indicated treatments (medium [M], 30 ng/ml TNF [T], 100 nM SMAC-mimetic [S], 20 µM z-VAD [Z]) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins
Techniques Used: Activation Assay, Transfection, SDS Page, Western Blot
![TNF treatment induces ATG14 and ATG16L1 puncta formation via RIPK3 and AMPK. ( A and B ) WT and ripk3 KO L929 cells were exposed to indicated treatments (medium [M], 10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM GSK’872 [G]) for 3 h. After that, cells were fixed and subjected to ATG14 (A) or ATG16L1 (B) immunostaining using anti-ATG14 (Santa Cruz Biotechnology, sc-164767) or anti-ATG16L1 antibodies (MBL International, PM040) and IRDye® 680RD donkey anti-goat or Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 120 (A) or 261 cells (B) was analyzed. ( C ) WT L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (si Prkaa1/ si Prkaa2 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 655 cells was analyzed. ( D ) WT L929 cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM AMPK inhibitor dorsomorphin) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 122 cells was analyzed. ( A-D ) Statistical analysis was performed using ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D; or two-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For B, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of WT vs. ripk3 KO cells). **** P < 0.0001. Scale bar: 20 µm](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6653/pmc08726653/pmc08726653__KAUP_A_1899667_F0002_C.jpg "... treatment induces ATG14 and ATG16L1 puncta formation via RIPK3 and AMPK. ( A and B ) WT ...")
Figure Legend Snippet: TNF treatment induces ATG14 and ATG16L1 puncta formation via RIPK3 and AMPK. ( A and B ) WT and ripk3 KO L929 cells were exposed to indicated treatments (medium [M], 10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM GSK’872 [G]) for 3 h. After that, cells were fixed and subjected to ATG14 (A) or ATG16L1 (B) immunostaining using anti-ATG14 (Santa Cruz Biotechnology, sc-164767) or anti-ATG16L1 antibodies (MBL International, PM040) and IRDye® 680RD donkey anti-goat or Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 120 (A) or 261 cells (B) was analyzed. ( C ) WT L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (si Prkaa1/ si Prkaa2 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 655 cells was analyzed. ( D ) WT L929 cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM AMPK inhibitor dorsomorphin) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 122 cells was analyzed. ( A-D ) Statistical analysis was performed using ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D; or two-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For B, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of WT vs. ripk3 KO cells). **** P < 0.0001. Scale bar: 20 µm
Techniques Used: Immunostaining, Software, Transfection
![RIPK3 interacts with AMPK. ( A ) S100 extracts of MEFs were separated by size-exclusion chromatography on a Superose 6 increase column. Fractions were analyzed by immunoblotting for the indicated proteins. The diagram shows protein levels for fractions 16–40 and the density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. ( B ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( C ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-AMPK antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( D ) L929 cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-IgG or anti-RIPK3 antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and RIPK3. ( E ) GST or GST-MmRIPK3 immobilized on glutathione-Sepharose beads was incubated with His-AMPK [His-HsPRKAA1 (11–559) + HsPRKAB2 (1–272) + HsPRKAG1 (1–331)] overnight. After washing the beads, bound proteins were eluted by boiling for 10 min at 95°C. Proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and GST. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-RIPK3: Prosci, 2283; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 216 cells was analyzed. Statistical analysis was performed using an unpaired t test with Welch’s correction. **** P < 0.0001. Scale bar: 20 µm](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6653/pmc08726653/pmc08726653__KAUP_A_1899667_F0003_C.jpg "RIPK3 interacts with AMPK. ( A ) S100 extracts ...")
Figure Legend Snippet: RIPK3 interacts with AMPK. ( A ) S100 extracts of MEFs were separated by size-exclusion chromatography on a Superose 6 increase column. Fractions were analyzed by immunoblotting for the indicated proteins. The diagram shows protein levels for fractions 16–40 and the density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. ( B ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( C ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-AMPK antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( D ) L929 cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-IgG or anti-RIPK3 antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and RIPK3. ( E ) GST or GST-MmRIPK3 immobilized on glutathione-Sepharose beads was incubated with His-AMPK [His-HsPRKAA1 (11–559) + HsPRKAB2 (1–272) + HsPRKAG1 (1–331)] overnight. After washing the beads, bound proteins were eluted by boiling for 10 min at 95°C. Proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and GST. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-RIPK3: Prosci, 2283; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 216 cells was analyzed. Statistical analysis was performed using an unpaired t test with Welch’s correction. **** P < 0.0001. Scale bar: 20 µm
Techniques Used: Size-exclusion Chromatography, Western Blot, Transfection, Plasmid Preparation, Immu-Puri, Purification, SDS Page, Incubation, Proximity Ligation Assay, Staining
![RIPK3 directly phosphorylates PRKAA1 at T183. ( A ) For in vitro kinase assay, purified GST, GST-HsPRKAA1(1–278) and GST-HsPRKAA1(279–559) were incubated with activated RIPK3 and [γ- 32 P]-ATP. The reactions were subjected to SDS-PAGE. After Coomassie Brilliant Blue staining and drying of the gels, autoradiography was performed. ( B ) GST-HsPRKAA1 WT and the T183A mutant were purified and were incubated with activated RIPK3 and cold ATP. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( C ) GST-HsPRKAA1 WT and the T183A mutant were incubated with activated RIPK3 and cold ATP with or without alkaline phosphatase. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( D ) GST-HsPRKAA1 WT was incubated with activated RIPK3 and cold ATP with or without 50 µM GSK’872. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( E ) HEK293 cells were left untransfected or were transfected with cDNA encoding either 3xFLAG-HsRIPK3 WT or 3xFLAG-HsRIPK3 kinase-dead (KD) for 24 h. After that, cells were treated with 30 ng/ml TNF + 30 µM QVD for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172, AMPK, FLAG and ACTB. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, the cells were left untreated (medium, M) or treated with 30 ng/ml TNF + 100 nM SMAC-mimetic + 20 µM z-VAD (TSZ) for 3 h. Then cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-phospho-PRKAA1/2 T183/T172: Cell Signaling Technology, 2535; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 107 cells was analyzed. Statistical analysis was performed using ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). **** P < 0.0001. Scale bar: 20 µm. ( G ) L929 cells were transiently transfected with cDNA encoding either 3xFLAG-HsPRKAG1 WT or R299G for 24 h. After that, cells were treated with or without 10 ng/ml TNF + 30 µM QVD (TQ) for 2 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6653/pmc08726653/pmc08726653__KAUP_A_1899667_F0004_C.jpg "RIPK3 directly phosphorylates PRKAA1 at T183. ( A ) ...")
Figure Legend Snippet: RIPK3 directly phosphorylates PRKAA1 at T183. ( A ) For in vitro kinase assay, purified GST, GST-HsPRKAA1(1–278) and GST-HsPRKAA1(279–559) were incubated with activated RIPK3 and [γ- 32 P]-ATP. The reactions were subjected to SDS-PAGE. After Coomassie Brilliant Blue staining and drying of the gels, autoradiography was performed. ( B ) GST-HsPRKAA1 WT and the T183A mutant were purified and were incubated with activated RIPK3 and cold ATP. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( C ) GST-HsPRKAA1 WT and the T183A mutant were incubated with activated RIPK3 and cold ATP with or without alkaline phosphatase. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( D ) GST-HsPRKAA1 WT was incubated with activated RIPK3 and cold ATP with or without 50 µM GSK’872. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( E ) HEK293 cells were left untransfected or were transfected with cDNA encoding either 3xFLAG-HsRIPK3 WT or 3xFLAG-HsRIPK3 kinase-dead (KD) for 24 h. After that, cells were treated with 30 ng/ml TNF + 30 µM QVD for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172, AMPK, FLAG and ACTB. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, the cells were left untreated (medium, M) or treated with 30 ng/ml TNF + 100 nM SMAC-mimetic + 20 µM z-VAD (TSZ) for 3 h. Then cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-phospho-PRKAA1/2 T183/T172: Cell Signaling Technology, 2535; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 107 cells was analyzed. Statistical analysis was performed using ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). **** P < 0.0001. Scale bar: 20 µm. ( G ) L929 cells were transiently transfected with cDNA encoding either 3xFLAG-HsPRKAG1 WT or R299G for 24 h. After that, cells were treated with or without 10 ng/ml TNF + 30 µM QVD (TQ) for 2 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins
Techniques Used: In Vitro, Kinase Assay, Purification, Incubation, SDS Page, Staining, Autoradiography, Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Proximity Ligation Assay, Immu-Puri
![Necroptosis inhibits lysosomal LC3 degradation. ( A ) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A 1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A 1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 4). ( B ) L929 WT, ripk3 KO or MLKL KO cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 3). ( C ) L929 cells retrovirally transfected with cDNA encoding mRFP-EGFP-rLC3 were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (si Ripk3 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 20 nM bafilomycin A 1 [B]) for 3 h. Then cells were fixed and RFP and GFP fluorescence was analyzed by immunofluorescence microscopy. The colocalization intensity was analyzed using Pearson’s correlation coefficient using ImageJ software. Scale bar: 20 µm. ( D ) L929 cells were retrovirally transfected with cDNA encoding mCitrine-LC3B. Cells were left untreated (medium, M) or treated using 10 ng/ml TNF + 30 µM QVD (TQ) or EBSS with or without 20 nM bafilomycin A 1 (B) for indicated times. Cells were collected and mCitrine fluorescence intensity was measured by flow cytometry. The mean of fluorescence intensity for each sample was normalized to cells incubated in growth medium (M). Data represent mean + SD from two independent experiments. ( A-D ) Statistical analysis was done by repeated measures two-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D, and by ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For C, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of non-targeting vs. Ripk3 siRNA). For A and B, statistically significant differences are only indicated for 6 h; for D, statistically significant differences are only indicated for 6 h vs. 6 h + bafilomycin A 1 . Statistically significant differences to control (medium, M) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6653/pmc08726653/pmc08726653__KAUP_A_1899667_F0005_C.jpg "... (n = 4). ( B ) L929 WT, ripk3 KO or MLKL KO cells were exposed to ...")
Figure Legend Snippet: Necroptosis inhibits lysosomal LC3 degradation. ( A ) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A 1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A 1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 4). ( B ) L929 WT, ripk3 KO or MLKL KO cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 3). ( C ) L929 cells retrovirally transfected with cDNA encoding mRFP-EGFP-rLC3 were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (si Ripk3 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 20 nM bafilomycin A 1 [B]) for 3 h. Then cells were fixed and RFP and GFP fluorescence was analyzed by immunofluorescence microscopy. The colocalization intensity was analyzed using Pearson’s correlation coefficient using ImageJ software. Scale bar: 20 µm. ( D ) L929 cells were retrovirally transfected with cDNA encoding mCitrine-LC3B. Cells were left untreated (medium, M) or treated using 10 ng/ml TNF + 30 µM QVD (TQ) or EBSS with or without 20 nM bafilomycin A 1 (B) for indicated times. Cells were collected and mCitrine fluorescence intensity was measured by flow cytometry. The mean of fluorescence intensity for each sample was normalized to cells incubated in growth medium (M). Data represent mean + SD from two independent experiments. ( A-D ) Statistical analysis was done by repeated measures two-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D, and by ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For C, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of non-targeting vs. Ripk3 siRNA). For A and B, statistically significant differences are only indicated for 6 h; for D, statistically significant differences are only indicated for 6 h vs. 6 h + bafilomycin A 1 . Statistically significant differences to control (medium, M) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001
Techniques Used: SDS Page, Western Blot, Transfection, Fluorescence, Immunofluorescence, Microscopy, Software, Flow Cytometry, Incubation
Figure Legend Snippet: Necroptosis induced by TNF destabilizes SNARE complexes and cleaves STX17 to block LC3 degradation. ( A ) L929 cells stably expressing GFP-SNAP29 were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. ( B ) L929 cells stably expressing GFP-SNAP29 were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ), TQ plus 5 µM GSK’872 (TQG), or TQ plus 5 µM necrostatin-1 (TQN) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. ( C ) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A 1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17 and ACTB. ( D ) L929 WT, ripk 3 KO or mlkl KO cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17, RIPK3, MLKL, and GAPDH. ( E ) L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (si Prkaa1/ si Prkaa2 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) with or without 5 µM GSK’872 (G) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (siCtrl, medium). Statistical graphics represents mean + SD (n = 3). Statistical analysis was done by ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). Statistically significant differences are only indicated for TQ and TQG (scr vs. Prkaa1/Prkaa2 siRNA) and for TQ vs. TQG (within each treatment). Statistically significant differences to control (siCtrl, medium) are depicted as letters directly above the bars. ** or b: P < 0.01, *** or c: P < 0.001, ns: non-significant. ( F ) L929 cells were left untransfected or transiently transfected with cDNA encoding 3xFLAG-MmRIPK3 for 24 h. Then untransfected cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 3 h. Cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins
Techniques Used: Blocking Assay, Stable Transfection, Expressing, Immu-Puri, Purification, SDS Page, Western Blot, Transfection
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Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse ripk3 - by Bioz Stars,
2023-06
94/100 stars
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1) Product Images from "A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression"
Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression
Journal: bioRxiv
doi: 10.1101/2021.02.14.431152
Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). (B) Cultured MCF7/TO-RIPK3, KGN/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with DMSO, Dox, or Dox plus TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. (C) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox, plus the indicated agents for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). 20 μM Z, pan-caspase inhibitor z-VAD; 2 μM RIPA-56, RIPK1 inhibitor; 2 μM NSA, MLKL inhibitor. (D) Cultured MCF7 cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(AAAA), RIPK3(K50A), and RIPK3(K50A)+GSK’872 plus Z for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). GSK’872, RIPK3 inhibitor. The online version of this article includes the following figure supplement(s) for : Figure supplement 1 . RIPK3-induced apoptosis in human granulosa lutein cells (KGN).
Techniques Used: Cell Culture, Western Blot, Infection
Figure Legend Snippet: Cultured KGN/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel).
Techniques Used: Cell Culture, Western Blot
Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus the indicated agent for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (B - E ) Cultured MCF7/TO-RIPK3(wild type (WT), RIPK1 -/- , Caspase8 -/- , FADD -/- , and cFLIF -/- ) cells were treated with DMSO or Dox induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK1, Caspase8, FADD, cFLIP or β-actin (lower panel).
Techniques Used: Cell Culture, Immunoprecipitation, Western Blot
Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The RIPK3 bands were excised and subjected to mass spectrometry analysis. RIPK3 specific phosphorylation site in MCF7/TO-RIPK3 cells is highlighted in red. (B) Cultured KGN/TO-RIPK3, MCF7/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (C) Cultured MCF7 stably transfected with either wild type RIPK3 (WT) or kinase-dead mutant (D160N) cells under the control of Dox-inducible promoter were treated with DMSO(-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured 293T cells were transfected with Vector (Vec), RIPK3(WT), RIPK3(D160N), RIPK3(AAAA) (RIPK3-AAAA, residues 459-462 mutated to AAAA) and RIPK3(S164D/T165E) for 24 hours. The level of phospho-S227-RIPK3 and RIPK3 were measured by western blotting. (E and F) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(S164D/T165E), RIPK3(S164A/T165A) and RIPK3(AAAA) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). (G) Cultured HeLa cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(D160N), RIPK3(AAAA), and RIPK3(S164D/T165E) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. The expressed RIPK3 in the cell lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). Vector (Vec, control viruses) (H) Cultured HeLa cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), and RIPK3(S164D/T165E) for 24 hours. RIPK3 was immunoprecipitated using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, Caspase-8, FADD, and RIPK3 as indicated. (I) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with Dox plus Z for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. The online version of this article includes the following figure supplement(s) for figure 3: Figure supplement 1 . Characterization of RIPK3 auto-phosphorylation sites. Figure supplement 2 . The phosphorylation site of RIPK3 is conserved among different mammalian species.
Techniques Used: Cell Culture, Immunoprecipitation, Mass Spectrometry, Western Blot, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation, Infection
Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (B) Cultured KGN7/TO-RIPK3 and KGN/TO-RIPK3(S164A/T165A) cells were treated with DMSO or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. ( C) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(K50A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured HeLa cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant RIPK3 including RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) and treated with TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. ** P <0.01. The levels of expressed RIPK3 in the cell lysates were measured by western blotting (lower panel). (E) RIPK3 single site (S164E) mutation blocks auto-phosphorylation. 293T cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) for 24 hours. The level of p-S227-RIPK3 and RIPK3 were measured by western blotting. (F and G) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant forms of RIPK3(S164A/T165A), RIPK3(S164A) and RIPK3(T165A) and treated with z-VAD as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The levels of RIPK3 in the cell lysates were measured by western blotting (lower panel). (H) Cultured HeLa cells were transfected with Flag-tagged wild type RIPK3(WT), and mutant forms of RIPK3(T165E), RIPK3(S164D/T165E), and RIPK3(S164E) for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, caspase-8, RIPK3, and β-actin as indicated.
Techniques Used: Cell Culture, Western Blot, Infection, Mutagenesis, Transfection, Immunoprecipitation
Figure Legend Snippet: (A) The cell lysates from cultured HT29, HeLa, MCF7, and KGN cells were analyzed by western blotting using antibodies as indicated. (B and C) Cultured HeLa-RIPK3, MCF7/TO-RIPK3, and KGN/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. 17AAG, Hsp90 inhibitor. (D and E) HeLa/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, and β-actin as indicated in (E). (F and G) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with DMSO or Dox for 36 hours. Cell viability was determined by measuring cellular ATP levels in (F). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, Hsp90, CDC37, and β-actin as indicated in (G). (H) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (I) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (J) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1 . Hsp90/CDC37 chaperone determines the necroptotic or apoptotic function of RIPK3 kinase. Figure supplement 2 . RIPK3 form amyloid-like structure in MCF7 and KGN cells. Figure supplement 3 . Hsp90/CDC37 chaperone protein levels was low in corpus luteum and corpus albicans.
Techniques Used: Cell Culture, Western Blot, Transfection, Immunoprecipitation, Immunofluorescence
Figure Legend Snippet: (A) HeLa/TO-RIPK3 and HeLa/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P <0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (B) L929 cells were treated with the indicated stimuli for 5 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. (C) L929( Ripk3 -/- )/TO-RIPK3 and L929( Ripk3 -/- )/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P < 0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (D and E ) L929( Ripk3 -/- )/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine165/Theronine166 of RIPK3, RIPK3, and β-actin as indicated in (E).
Techniques Used: shRNA, Western Blot
Figure Legend Snippet: (A) Alignment of amino acid sequences of RIPK3 orthologs in five mammalian species. Amino acid residues conserved in 80% or more of the sequences are shaded in black. The putative phosphorylation residues are denoted by asterisks (*). (B) Cultured 293T cells were transfected with Vector (Vec), mouse RIPK3(WT), RIPK3(S165D/T166E), and RIPK3(S165A/T165A) for 24 hours. The level of phospho-S232-RIPK3 and RIPK3 were measured by western blotting. (C) Cultured mouse sarcoma cells L929( Ripk3 -/- ) were transfected with Vector, wild type mouse RIPK3, and mutant forms of mRIPK3(D161N), and mRIPK3(S165D/T166E) and treated with z-VAD or TSZ as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. The lysates were analyzed by western blotting using antibodies as indicated (lower panel).
Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Western Blot, Mutagenesis
Figure Legend Snippet: (A) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus TSZ for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (B) Cultured HT29 and KGN/TO-RIPK3 cells were treated with DMSO, TSZ, or Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (C) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (D) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (E) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with DMSO or Dox plus Z for 24 hours. The lysates were analyzed by western blotting using antibodies as indicated.
Techniques Used: Cell Culture, Immunofluorescence, Western Blot
Figure Legend Snippet: (A) Two guide RNA and donate oligo sequences of Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. (B) Schematic of CRISPER-Cas9 strategy for the generation for Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. The gene structure of RIPK3 and two guide RNA sequences targeting the exon 4 of RIPK3 were shown with the PAM sequences highlighted in red and blue. (C and D ) Macroscopic features (C) and body weights (D) of Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice at 14 days of age (n=10). The result from each individual animal was presented as an indicated dot. NS, not significant. (E and F ) Immunoblot of RIPK3 from lung extracts of 14 days old Ripk3 +/+ , Ripk3 ST-DE/ST-DE , and Ripk3 ST-AA/ST-AA littermates using antibodies against RIPK3 and GAPDH as indicated (n=3). (G) Histological analysis of brain, cerebellum, heart, kidney, and liver of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. PTC, Proximal tubular cell. (H) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice (n=3, 14 days) after treatment with the indicated necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells.
Techniques Used: Knock-In, Western Blot, Derivative Assay
Figure Legend Snippet: (A and B ) Macroscopic features (A) and body weights (B) of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice at 14 days of age (n≥9). The result from each individual animal is presented as an indicated dot. *** P <0.001. (C) Kaplan-Meier plot of survival of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=10 for each genotype) after birth within two months. *** P <0.001. (D) Histological analysis of large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. (E and F) Representative immunohistochemistry (IHC) images of the large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5, 14 days) stained with a Cleaved-Caspase3 (C-C3) antibody in (E). C-C3 positive cells were counted in two fields per organ and quantified in (F). Scale bar, 10 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (G) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=3, 14 days) after treatment with the indicated Z-VAD or necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. The online version of this article includes the following figure supplement(s) for figure 5: Figure supplement 1 . Generation of Ripk3 ST-DE/ST-DE and Ripk3 ST-AA/ST-AA mice.
Techniques Used: Immunohistochemistry, Staining, Derivative Assay
Figure Legend Snippet: (A) Western blot analysis of RIPK1, RIPK3, and MLKL levels in perfused mouse ovary extracts of different ages. Each group is representative of at least 3 mice. (B) H&E and immunofluorescence (IF) imaging of an 8-month-old ovary. Two adjacent sections were analyzed. One section was stained with H&E, and the other was IF stained with a RIPK3 antibody (red) and DAPI (blue). Scale bar, 500 μm. Higher-power views of selected areas were acquired in a (primordia follicle), b (secondary follicle), c (corpus luteum), and d (corpus albicans) as indicated. PF, primary follicle; CL, corpus luteum; CA, corpus albicans. (C) Ovarian PGF 2α levels of wild-type mice (n=8) at the indicated age assayed by ELISA. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (D) Immunofluorescence images of a RIPK3 C-terminus HA-3×Flag knock-in mouse ovary (n=5; 12 months) stained with antibodies against prostaglandin F receptor (PTGFR, green) and Flag (red). Counterstaining with DAPI (blue). Scale bar, 500 μm. Higher-power views (right panels) were acquired from the indicated boxed area in the second lower left panel. CL, corpus luteum. CA, corpus albicans. Scale bar, 100 μm. (E) Western blot analysis of p-S164/T165-RIPK3 and RIPK3 levels in extracts from perfused ovaries prepared from mice at the indicated age. Each group is representative of at least 3 mice. (F) Immunofluorescence images of ovaries from Ripk3 +/+ and Ripk3 -/- mice (4 Month, 8 Month and 12 Month; n=3) at the indicated ages stained with the p-S164/T165-RIPK3 antibody (red). Counterstaining with DAPI (blue). Scale bar, 200 μm. Higher-power views (lower two panels) were acquired from the selected boxed areas from the upper panel. (CL), b (CL), c (CL, CA) and d (CL). F, follicle; CL, corpus luteum; CA, corpus albicans. Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 6: Figure supplement 1 . Generation of RIPK3 C-terminus HA-3×Flag knock-in and Ptgfr -/- mice.
Techniques Used: Western Blot, Immunofluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Knock-In
Figure Legend Snippet: (A) Schematic of CRISPER-Cas9 strategy for RIPK3 C-terminus HA-3×Flag knock-in mice. The gene structure of RIPK3 and guide RNA sequences targeting the Ripk3 were shown with the PAM sequences highlighted in red. (B) Western blotting analysis using protein extracts from the ovary of wild type, heterozygous knock-in, and homozygous knock-in mice generated as illustrated in (A). (C) Schematic of CRISPER-Cas9 strategy for the generation for Ptgfr -/- mice. The gene structure of PTGFR and two guide RNA sequences targeting the Ptgfr were shown with the PAM sequences highlighted in red. (D) Immunoblot of PTGFR from ovary extracts of 2-month old Ptgfr +/+ and Ptgfr -/- littermates using antibodies against PTGFR and GAPDH as indicated (n=3).
Techniques Used: Knock-In, Western Blot, Generated
Figure Legend Snippet: (A and B ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and HSP90 (green) antibody in (A). Higher-power views of selected areas were acquired in right panel. The HSP90/RIPK3 levels were quantified in (B). F, follicle. CA, corpus albicans. Scale bar, 100/200 μm. ( C and D ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and CDC37 (green) antibody in (C). Higher-power views of selected areas were acquired in right panel. The CDC37/RIPK3 levels were quantified in (D). F, follicle. CA, corpus albicans. Scale bar, 200 μm.
Techniques Used: Immunofluorescence
Figure Legend Snippet: (A to C) Primary granulosal lutein cells (WT, Ripk3 -/- ) were isolated from 3-month-old mice ovaries. The cells were treated with Dinoprost Tromethamine (DT) at the indicated concentration for 36 hours in (A); with 1.5 μM DT at the indicated time in (B); or with 1.5 μM DT for 36 hours in (C). The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (D and E) Ptgfr +/+ and Ptgfr -/- littermate female mice (n=16; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. Ovaries were then collected 12 hours later and stained with anti-RIPK3 antibody (red) in (D). The ovary lysates were analyzed by western blotting using antibodies as indicated in (E). (*) indicates Corpus luteum. Counterstaining with DAPI (blue). Scale bar, 500 μm. (F and G) wild type(WT), Ripk3 -/- , Ripk3 S165A-T166A/S165A-T166A , Fadd -/- Mlkl -/- and Ptgfr -/- female mice (each group, n=16; 25-26 days) were treated as in (D and F). Ovaries from each group were then collected 24 hours after injecting with DT and stained with anti-cleaved-caspase3 antibody in (F). The Cleaved-Caspase3 + cells were counted in five fields per ovary Corpus luteum(CL) and quantified in (G). Scale bar, 20 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (H) wild type female mice (n=3; 25-26 days) were treated as in (D and F). Ovaries were then collected 12 hours after injecting with DT and stained with anti-cleaved-caspase3(red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 7: Figure supplement 1 . Prostaglandin F2alpha (PGF 2α ) stimulates RIPK3 expression through the MAPK pathway.
Techniques Used: Isolation, Concentration Assay, Western Blot, Injection, Staining, Expressing
Figure Legend Snippet: (A) Primary granulosal lutein cells were isolated from the 3-month-old mice ovary. The cells were then treated with 1 μM DT or plus MAPK inhibitors PD-98059 (5 μM) and U0126 (5 μM) as indicated for 36 hours. The lysates were analyzed by western blotting using antibodies as indicated. (B) Primary granulosal lutein cells were isolated from 3-month-old mice ovaries. The cells were treated with 1 μM DT at the indicated time. The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (C) Diagram of induction of corpus luteum regression in vivo . (D) Ripk3 +/+ and Ripk3 S165A-T166A/S165A-T166A and littermate female mice (n=3; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. The ovary lysates were analyzed by western blotting using antibodies as indicated.
Techniques Used: Isolation, Western Blot, In Vivo, Injection
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Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse rip3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse rip3 - by Bioz Stars,
2023-06
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1) Product Images from "Casein Kinase 1G2 Suppresses Necroptosis-Promoted Testis Aging by Inhibiting Receptor-Interacting Kinase 3"
Article Title: Casein Kinase 1G2 Suppresses Necroptosis-Promoted Testis Aging by Inhibiting Receptor-Interacting Kinase 3
Journal: bioRxiv
doi: 10.1101/2020.08.17.254318
Figure Legend Snippet: (A) CSNK1s associate with RIP3. The HT29-HA-3×Flag-RIP3 cell lysates were immunoprecipitated using anti-Flag resin. The pull-down protein mixture was subjected to mass spectrometry analysis and the identify casein kinase were shown. (B) Measurement of the effect of co-expressed casein kinase members on RIP3 kinase activity. Cultured 293T cells were co-transfected with Flag-tagged RIP3 and indicated Myc-tagged CSNK1A1, CSNK1A1-L, CSNK1D1, CSNK1D2, CSNK1G1, CSNK1G2, CSNK1E, CSNK2A1, CSNK2A2 and CSNK2B for 20 hrs. The cell extracts were then subjected to western blotting analysis using antibodies against Myc-tag, Flag-tag, β-actin, and phosphor-S227-RIP3 as indicated. Numbers on the right indicate molecular weight markers (kDa). (C) Measurement of the effect of co-expression casein kinase 1G members on RIP3 kinase activity. Cultured 293T cells were co-transfected with Flag-tagged RIP3 and indicated Myc-tagged CSNK1G1, CSNK1G2, and CSNK1G3 for 20 hrs. The cell extracts were then subjected to western blotting analysis using antibodies against Myc-tag, Flag-tag, β-actin, and phosphor-S227-RIP3 as indicated.
Techniques Used: Immunoprecipitation, Mass Spectrometry, Activity Assay, Cell Culture, Transfection, Western Blot, FLAG-tag, Molecular Weight, Expressing
Figure Legend Snippet: (A) Western blotting analysis using antibodies against the indicated proteins. Cultured 293T cells were transfected with Flag-tagged RIP3 and the indicated versions of Myc-tagged CSNK1G2, including wild type (WT) and two kinase-dead point mutants K75A and D165N for 20 hrs. Cell extracts were then prepared and used for western blotting analysis. Vec, vector control. Numbers on the right indicate molecular weight markers (kDa). (B) Top: Cell viability as measured by Cell Titer-Glo. Cultured MEF with wild type CSNK1G2 gene (WT) or with their CSNK1G gene knocked out (KO) followed by transfection with vector control (Vec) or indicated wild type or a kinase-dead (K75A) mutant CSNK1G2 MEF. The cells were then treated with DMSO or TSZ as indicated for 12 hrs before the intracellular ATP levels were measured by Cell Titer-Glo. T denotes 20 ng ml -1 TNF-α; S, denotes 100 nM Smac mimetic; Z denotes 20 μM Z-VAD-FMK. Data are mean ± SD of triplicate wells. *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. Bottom: Aliquots of these treated cells were used for western blotting analysis using an antibody against CSNK1G2. (C) Western blotting of necroptosis activation markers phospho-RIP3 (p-RIP3) and phospho-MLKL (p-MLKL). Cultured MEF cells with indicated CSNK1G2 gene as in (B) were treated with indicated stimuli for 4 hrs before the cell extracts were prepared and subjected to western blotting analysis as indicated. (D and E) Western blotting analysis of RIP3-associated RIP1 and CSNK1G2. Immuno-precipitates using an anti-Flag antibody from extracts of MEF-Flag-RIP3 and MEF ( CSNK1G2 -/- )-Flag-RIP3 cells (D) or HeLa-HA-3×Flag-RIP3-Myc-CSNK1G2(WT and K75A) cells (E) treated with the indicated stimuli for 6 hrs were subjected to western blotting analyzing using antibodies as indicated. (F) Cell viability measurement of bone marrow-derived macrophages from the wild type or CSNK1G2 knockout mice. Macrophages were isolated from the wild type (WT) or CSNK1G2 knockout mice (KO) and treated with the indicated necroptosis stimuli for 12 hrs, and the cell viability was measured by Cell-Titer Glo. Trail: TNF-related apoptosis-inducing ligand. LPS: Lipopolysaccharide. Data are mean ± SD of triplicate wells. *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. (G) Kaplan-Meier plot of survival of male CSNK1G2 +/+ (wild-type), CSNK1G2 -/- ( CSNK1G2 knockout littermates), or RIPK3 -/- (RIP3 gene knockout) mice (n=10 for each genotype, age: 3 months) injected intraperitoneally with one dose of murine TNF-α (300 μg kg -1 ). Body temperature was measured with a lubricated rectal thermometer. Mice with a temperature below 23 °C were euthanized for ethical reasons. See also , and .
Techniques Used: Western Blot, Cell Culture, Transfection, Plasmid Preparation, Molecular Weight, Mutagenesis, Activation Assay, Derivative Assay, Knock-Out, Isolation, Gene Knockout, Injection
Figure Legend Snippet: (A and B) The necroptotic effect of knockout CSNK1G2 in MEFs. Cultured parental MEF cells (WT) and MEF cells with their CSNK1G2 knocked out (KO) were treated with the indicated necroptotic stimuli for 12 hrs. The cell viability of these necroptotic stimuli-treated cells was then measured using Cell-titer Glo in (A). Data are mean ± SD of triplicate wells. *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. LPS 2 ng ml -1 , TRAIL 20 ng ml -1 . The cell extracts were then subjected to western blotting analysis using antibodies against RIP1, phosphor-S227-RIP3 (p-RIP3), β-actin, MLKL, and phosphor-S345-MLKL as indicated in (B). (C) Cultured MEF cells with their CSNK1G2 gene knocked out ( CSNK1G2 -/- ) were co-transfected with cDNAs encoding Myc-tagged wild type CSNK1G2 (WT) or kinase-dead mutant (K75A) as indicated and Flag-RIP3 for 24 hrs. The cell extracts were then subjected to western blotting analysis directly (Input), or immunoprecipitation with anti-Flag antibody and the precipitates were subjected to western blotting analysis using antibodies against Myc-tag, Flag-tag, and β-actin as indicated. (D) The effect of wild type and kinase-dead mutant of CSNK1G2 on RIP3 dimerization induced necroptosis. Cultured NIH3T3-Flag-RIP3-Myc-CSNK1G2(Vec, WT, and K75A) cells were treated with the FKBP dimerizer molecule AP20187 for 12h. The cell extracts were subsequently subjected to western blotting analysis using antibodies against Myc-tag, Flag-tag, and β-actin as indicated (lower panel). The cell viability was measured by Cell-titer Glo. Data are mean ± SD of triplicate wells.
Techniques Used: Knock-Out, Cell Culture, Western Blot, Transfection, Mutagenesis, Immunoprecipitation, FLAG-tag
Figure Legend Snippet: (A) MS/MS spectrum of CSNK1G2 phosphorylation sites. The identified phosphorylated peptide to be EHK P SLTG P TAR with S211 and T215 as the phosphorylated amino acid residues. The b and y type product ions are indicated in the spectrum. Data are related to those in . (B) Effect on RIP3 auto-phosphorylation by co-expression of the indicated version of CSNK1G2. Cultured 293T cells were transfected with Flag-tagged RIP3 cDNA together with indicated Myc-tagged wild type CSNK1G2 (WT), or kinase-dead mutant K75A, or phosphorylation site resistant mutant S211A/T215A for 20 hrs. The cell extracts were then subjected to western blotting analysis using antibodies against phospho-S227-RIP3, Flag-RIP3, and Myc-CSNK1G2 as indicated. (C) Top: Cell viability measurement of effect of phosphorylation sites mutant CSNK1G2 on necroptosis. Cultured wild type MEF (WT), or MEF with their CSNK1G gene knocked out (KO) were transfected with either vector control (Vec) or cDNA encoding wild type CSNK1G2 (WT) or phosphorylation sites mutant (S211A/T215A) followed by treatment of DMSO or necroptotic stimuli TSZ as indicated for 12 hrs. The cell viability was measured by Cell-titer Glo. Data are mean ± SD of triplicate wells. *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. Bottom: Aliquots of these treated cells were used to make cell extracts for western blotting analysis using an antibody against CSNK1G2 protein. (D) The effect of CSNK1G2 on RIP1/RIP3 interaction as measured by co-IP. Cultured HeLa-HA-3×Flag-RIP3 cells were transfected with either vector control (Vec) or wild type (WT) or phosphorylation site mutant Myc-CSNK1G2 (S211A/T215A) as indicated. The cells were then treated with DMSO or necroptosis stimuli TSZ for 6 hrs. The cell extracts were prepared and subjected to immunoprecipitation with an anti-Flag antibody. The extracts (Input) and the immuno-precipitates (IP: Flag) were then subjected to western blotting analysis using antibodies as indicated. See also .
Techniques Used: Tandem Mass Spectroscopy, Expressing, Cell Culture, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation
Figure Legend Snippet: (A) Identification of auto-phosphorylation sites on CSNK1G2. Myc-tagged wild type CSNK1G2 or its kinase-dead mutant (WT, K75A) were co-transfected with Flag-RIP3 in 293T cells for 24 hrs. CSNK1G2 was immunoprecipitated using anti-Myc resins. The CSNK1G2 (WT, K75A) bands were excised and analyzed by MS/MS. The identified phosphorylated peptides were shown in the table with the phosphorylated amino acid residues highlighted in red. No phosphorylated peptide was identified in CSNK1G2 (K75A) sample. (B) The effect of phosphorylation sites mutants CSNK1G2 on RIP3 kinase activity. Cultured 293T cells were transfected with vector control (Vec) or Flag-tagged RIP3 and Myc-tagged wild type (WT), or kinase-dead (K75A) or different phosphorylation site mutants as indicated (S26A/S27A, S211A/T215A and S381A) for 20 hrs. The cell extracts were then subjected to western blotting analysis using antibodies against Flag-tag, Myc-tag, phosphor-Serine227-RIP3 and β-actin as indicated. (C) The effect of S211A and T215A mutants of CSNK1G2 on RIP3 kinase activity Cultured 293T cells were transfected with vector control (Vec) or Flag-tagged RIP3 and Myc-tagged wild type (WT), or a kinase-dead mutant (K75A), or S211A or T215A mutants for 20 hrs. The cell extracts were then subjected to western blotting analysis using antibodies against Flag-tag, Myc-tag, phosphor-Serine227-RIP3 and β-actin as indicated. (D) Alignment of amino acid sequences around the auto-phosphorylation sites of CSNK1G2 in four vertebrate species. The Serine 211 and Threonine 215 (human origin) residues are denoted by asterisks (*). The numbers on the right indicate the corresponding histidine in CSNK1G2 of indicated species.
Techniques Used: Mutagenesis, Transfection, Immunoprecipitation, Tandem Mass Spectroscopy, Activity Assay, Cell Culture, Plasmid Preparation, Western Blot, FLAG-tag
Figure Legend Snippet: (A) The expression of CSNK1G2 in mouse testis, lung, large intestine, small intestine, spleen, kidney, heart, brain, liver and ovary tissues (n=3, 2 months). The indicated tissue extracts were subjected to western blotting analysis using antibodies against CSNK1G2 and GAPDH as indicated. (B) The expression of RIP3 and CSNK1G2 in mouse testis. The testis sections of 2-month old wild type mice (n=3) were stained sequentially with antibodies against RIP3 and CSNK1G2 followed by fluorescent-conjugated secondary antibody. Counterstaining with DAPI, blue. Scale bar on the upper panel is 100 μm. The areas marked by the yellow boxes on the upper panel were shown in the lower panel. (C) The sensitivity of primary cells from the seminiferous tubules of CSNK1G2 -/- and CSNK1G2 +/+ testis to necroptosis induction. The cells from the seminiferous tubules of 2-month old littermates with indicated genotype were isolated and cultured in vitro before treated with DMSO or TSZ as indicated for 12 hrs. The cell viability was then measured by Cell-Titer Glo. Data are mean ± SD of triplicate wells. *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. (D) The expression of RIP3, CSNK1G2 and RIP1 protein in GC-2spd, 15P-1 and MA-10 cells. The extracts from the indicated cultured cells were subjected to western blotting analysis using antibodies against RIP3, CSNK1G2, RIP1 and GAPDH as indicated. (E) Immunofluorescent analysis of RIP3 and CSNK1G2 expression in GC-2spd, 15P-1, and MA-10 cells. The GC-2spd, 15P-1, and MA-10 cells cultured on cover slides were sequentially stained with antibodies against RIP3 and CSNK1G2 followed by secondary antibodies conjugated with red (RIP3) or green (CSNK1G2). Counterstaining with DAPI, blue. Scale bar, 10 μm. (F and G) The effect of CSNK1G2 on necroptosis of GC-2spd and 15P-1 cells. Cultured parental GC-2spd (f) or 15P-1 cells (g) (WT), and GC-2spd or 15P-1 cells with their CSNK1G2 gene knocked out ( CSNK1G2 -/- ) were treated with DMSO or TSZ as indicated for 4 hrs. The cell viability was measured by Cell-titer Glo. Data are mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. Bottom, Immunoblot of CSNK1G2. Cell extracts from aliquots of these cells were also subjected to western blotting analysis using antibodies against CSNK1G2 and GAPDH as indicated, and the results were shown in the bottom.
Techniques Used: Expressing, Western Blot, Staining, Isolation, Cell Culture, In Vitro
Figure Legend Snippet: (A) Body weights of CSNK1G2 +/+ and CSNK1G2 -/- male littermate mice when they were 2- and 12-months old (n=10 for each genotype). (B and C) Macroscopic features (B) and weights (C) of seminal vesicles from CSNK1G2 +/+ and CSNK1G2 -/- male littermate mice (n=10 for each genotype) at the indicated ages. (D and E) Macroscopic features (D) and weights (E) of testes from CSNK1G2 +/+ and CSNK1G2 -/- male littermate mice (n=12 for each genotype) at the indicated ages. (F) H&E staining sections of testis from CSNK1G2 +/+ and CSNK1G2 -/- male littermate mice (n=10 for each genotype) at the indicated ages. The number of empty seminiferous tubules was counted based on H&E staining, and the percentage of empty seminiferous tubules of each group is labeled in the upper left corner of the images. Scale bar, 200 μm. (G and H) Immunohistochemical staining (IHC) of testes from CSNK1G2 +/+ and CSNK1G2 -/- male littermate mice (n=6 for each genotype) with phospho-MLKL (p-MLKL) antibody in (G). p-MLKL positive cells were counted in five fields per testis and quantified in (H). Scale bar, 100 μm. (I) Western blotting analysis of extracts from phosphate-buffered saline(PBS) perfused testes of CSNK1G2 +/+ (WT) and CSNK1G2 -/- (KO) male littermate mice of 2- and 12-month of age using antibodies against CSNK1G2, RIP1, RIP3, MLKL and phospho-MLKL (p-MLKL) and β-actin as indicated. The number on the right is markers of molecular weight (kDa). Each group was from a pool of three mice. (J) Summary of fertility rates of CSNK1G2 +/+ and CSNK1G2 -/- male littermate mice (n=12). Each male mice of 2- or 12-month of age was housed in the same cage with a pairs of 10-week-old wild-type female mice for 3 months; females were replaced every 2 weeks. The number of male mice with reproduction capacity was counted. P values were determined using Fisher’s exact tests (unpaired, two-tailed). All quantified data in the figure except (J) represent the mean ± s.e.m. * P <0.05, ** P <0.01, *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. NS, not significant.
Techniques Used: Staining, Labeling, Immunohistochemical staining, Western Blot, Molecular Weight, Two Tailed Test
Figure Legend Snippet: (A) The body weights of 12-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- fed with a RIP1 kinase inhibitor (RIPA-56)-containing diet, and CSNK1G2 -/- RIP3 -/- male littermate mice (n=10 for each genotype). (B) The weights of seminal vesicles from 12-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56, and CSNK1G2 -/- RIP3 -/- male littermate mice (n=10 for each genotype). (C) The weights of testes from 12-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56, and CSNK1G2 -/- RIP3 -/- male littermate mice (n=10 for each genotype). (D) H&E staining of testis sections from 12-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56, and CSNK1G2 -/- RIP3 -/- male littermate mice (n=10 for each genotype). The number of empty seminiferous tubules was counted based on H&E staining, and the percentage of empty seminiferous tubules was labeled in the upper left corner of the images: scale bar, 200 μm. (E and F) IHC staining of testes from 12-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56 and CSNK1G2 -/- RIP3 -/- male littermate mice (n=8 for each genotype) with an anti-phospho-MLKL (p-MLKL) antibody (E). p-MLKL positive cells were counted in five fields per testis and quantified in (F). Scale bar, 100 μm. (G) Summary of the fertility rates of 12-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56, and CSNK1G2 -/- RIP3 -/- male littermate mice (n=10 for each genotype). Each male mouse was caged with a pair of 10-week-old wild-type female mice for 3 months; females were replaced every 2 weeks. The number of male mice with reproduction capacity was counted. P values were determined using Fisher’s exact tests (unpaired, two-tailed). CSNK1G2 -/- +RIPA-56 mice: CSNK1G2 -/- male mice were fed with AIN93G or AING3G containing RIPA-56 (RIPA-56: 300 mg kg -1 ) for 10 months started when they were 2-month old in an SPF facility. All quantified data in the figure except (G) represent the mean ± s.e.m. ** P <0.01, *** P <0.001. P values were determined by two-sided unpaired Student’s t tests. NS, not significant. See also and .
Techniques Used: Staining, Labeling, Immunohistochemistry, Two Tailed Test
Figure Legend Snippet: (A) Macroscopic features of a typical 12-month old seminal vesicle from CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56, and CSNK1G2 -/- RIP3 -/- male littermate mice (n=10 for each genotype examined). (B) Macroscopic features of a typical 12-month testes from CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56 and CSNK1G2 -/- RIP3 -/- male littermate mice (n=10 for each genotype examined). (C) Body weights of 3-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56 and CSNK1G2 -/- RIP3 -/- male mice (n=10 for each genotype). P values were determined by two-sided unpaired Student’s t-tests. NS, not significant.
Techniques Used:
Figure Legend Snippet: (A-D) Serum hormonal levels of 3-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56 and CSNK1G2 -/- RIP3 -/- male littermate mice. Littermates male mice with the indicated genotype were sacrificed, and the indicated hormone levels in serum from were measured using ELISA kit for each hormone (n=8 for each genotype). Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. P values were determined by two-sided unpaired Student’s t-tests. CSNK1G2 -/- +RIPA-56 mice: 2-month-old CSNK1G2 -/- male mice were fed with AIN93G or AING3G containing RIPA-56 (RIPA-56: 300 mg kg -1 ) for 1 months in an SPF facility before used. (E-H) Serum hormonal levels of 12-month old CSNK1G2 +/+ , CSNK1G2 -/- , CSNK1G2 -/- +RIPA-56 and CSNK1G2 -/- RIP3 -/- male littermate mice. Littermates of 12-month old male mice with the indicated genotype were sacrificed, and the indicated hormone levels in serum from were measured using ELISA kit for each hormone (n=8 for each genotype). Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. P values were determined by two-sided unpaired Student’s t-tests. CSNK1G2 -/- +RIPA-56 mice: 2-month-old CSNK1G2 -/- male mice were fed with AIN93G or AING3G containing RIPA-56 (RIPA-56: 300 mg kg -1 ) for 10 months in an SPF facility.
Techniques Used: Enzyme-linked Immunosorbent Assay
Figure Legend Snippet: (A) Expression of CSNK1G2 and RIP3 in human testes. Sections from a testis sample of a 30-years old human patient were stained sequentially with antibodies against CSNK1G2 and RIP3 as indicated followed by green or red fluorescent-conjugated secondary antibodies as indicated. Counterstaining with DAPI, blue. Scale bar, 50/100 μm. Yellow boxes in the upper panels were shown in the lower panels. The experiment was repeated three times with three different patients. (B and C) H&E staining of testes from young and old man. Young man testes (25-30 years, n=10; from testicular torsion necrosis patients) and old man testes (80-89 years, n=15; from prostate cancer patients) were sectioned and stained with H&E in (B). The number of empty seminiferous tubules was counted based on H&E staining and quantification in (C), empty seminiferous tubules were counted in five fields per testis. Scale bar, 100μm. (D and E) IHC of testes from young and old man with phosphor-Serine358-MLKL antibody (p-MLKL). Young man testes (25-30 years, n=10; from testicular torsion necrosis patients) and old man testes (80-89 years, n=15; from prostate cancer patients) were sectioned and stained with an antibody against phosphor-Serine358-MLKL antibody (D). p-MLKL + cells were counted in five fields per testis and quantification in (E). Scale bar, 100μm. All quantified data in the figure represent the mean ± s.e.m. *** P <0.001. P values were determined by two-sided unpaired Student’s t tests.
Techniques Used: Expressing, Staining
mouse ripk3 (ProSci Incorporated)
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mouse ripk3
Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse ripk3 - by Bioz Stars,
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anti mouse ripk3 (ProSci Incorporated)
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ProSci Incorporated manufactures this product
94
Structured Review
ProSci Incorporated
anti mouse ripk3
Anti Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Anti Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
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anti mouse ripk3 - by Bioz Stars,
2023-06
94/100 stars
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1) Product Images from "Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation"
Article Title: Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation
Journal: Cell Death & Disease
doi: 10.1038/s41419-020-2534-9
Figure Legend Snippet: a DAPK1 deficiency does not affect expressions of FADD, RIPK1, RIPK3, or MLKL in BMDMs. b , c Dapk1 − /− BMDMs exhibit increased cell death induction relative to WT upon zVAD+AT-406 treatment. WT and Dapk1 −/− BMDMs were stimulated with DMSO, AT-406 (0.6 μM, A), zVAD (20 μM, Z), Nec-1 (40 μM, N), or BV6 (0.5 μM, B), as indicated, for 18–20 h, before determining cell death according to release of ATP. d , e zVAD+TNF or zVAD+IFN-β treatments trigger increased necroptosis in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD + TNF (5 ng/ml) ( d ) or zVAD + IFN-β (5 ng/ml) ( e ) and then cell viability was determined. f High dose of AT-406 induces necroptosis. WT and Dapk1 −/− BMDMs were treated with AT-406 at the indicated dose, without or with Nec-1, and cell viability quantitated. Values are mean ± SD of triplicates in a single experiment. * P < 0.05, ** P < 0.01, *** P < 0.001 for unpaired t -test. Data have been repeated in two ( e , f) or three ( a – d ) independent experiments.
Techniques Used:
Figure Legend Snippet: a Knockdown of DAPK1 in HT-29 cells. HT-29 cells were transduced with pLL3.7-shCtrl or pLL3.7-shDAPK1, sorted, and then expressions of DAPK1, RIPK1 and RIPK3 were determined. b Increased zVAD+BV6-induced necroptosis in DAPK1-deficient HT-29 cells. Control and DAPK1-knockdown HT-29 cells were treated with zVAD (20 μM), BV6 (1 μM), Nec-1 (40 μM) as indicated for 24 h, cell death was quantitated by PI staining. Values are mean ± SD of triplicates in a single experiment. *** P < 0.001 for unpaired t -test. Results have been confirmed in three independent experiments. c Enhanced zVAD+BV6-induced phosphorylation of RIPK1, RIPK3 and MLKL in DAPK1-deficient HT-29 cells. Control and DAPK1-knockdown HT-29 cells were treated with zVAD+BV6 (0.5 μM, Z + B) and contents of RIPK1, pS166-RIPK1, RIPK3, pS227-RIPK3, MLKL, and pMLKL in cell lysates were determined at the indicated time points. Right panel, quantitation of pRIPK1(S166), pRIPK3 and pMLKL from three independent experiments using normalized intensity of pRIPK1(S166), pRIPK3 and pMLKL in DAPK1-knockdown HT29 cells at 9 h as 1. ** P < 0.01, *** P < 0.001 for two-way ANOVA followed by a Tukey’s multiple comparison test.
Techniques Used: Transduction, Staining, Quantitation Assay
Figure Legend Snippet: a , b Increased zVAD+AT-406-induced phosphorylation of RIPK1, RIPK3 and MLKL in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD (20 μM) plus AT-406 (0.6 μM, Z + A), and cell lysates were prepared at the indicated time points. The contents of RIPK1, pS166-RIPK1 ( a ), or RIPK3, pRIPK3, MLKL, and pMLKL ( b ) were determined by Western blot. Right panel, quantitation of pRIPK1(S166) ( a ) and pMLKL ( b ) from three independent experiments using normalized intensity of pRIPK1(S166) (3 h) and pMLKL (6 h) in Dapk1 −/− BMDMs as 1. *** P < 0.001 for two-way ANOVA followed by a Tukey’s multiple comparison test. c , d Increased activation of MLKL in Dapk1 −/− BMDMs stimulated by treatment with zVAD plus TNF or IFN-β. WT and Dapk1 −/− BMDMs were treated with zVAD in combination with TNF ( c ), or IFN-β ( d ) and levels of MLKL and pMLKL in cell lysates were determined at the indicated time points. Data are representative of three independent experiments.
Techniques Used: Western Blot, Quantitation Assay, Activation Assay
Figure Legend Snippet: a , b Enhanced zVAD+AT-406-induced binding of RIPK1 and RIPK3 to FADD/caspase-8 in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD+AT-406 (Z + A), and whole-cell lysates (WCL) were prepared at the indicated time points. WCL were immunoprecipitated with anti-FADD and contents of pRIPK1, RIPK1 and FADD in the precipitates and WCL were determined ( a ), or they were immunoprecipitated with anti-caspase-8 and the amounts of RIPK1, RIPK3, FADD and caspase-8 in the precipitates and WCL were determined ( b ). c , d Increased binding of RIPK1 and RIPK3 to FADD/caspase-8 in DAPK1-deficient HT-29 cells upon necroptotic induction. Control and DAPK1-knockdown HT-29 cells were treated with zVAD+BV6 + TNF (Z + B + T) ( c ) or zVAD+BV6 (Z + B) ( d ) before collecting WCL at the indicated time points. WCL were immunoprecipitated with anti-FADD and the contents of pRIPK1, RIPK1 and FADD in the precipitates and WCL were determined (c ), or we immunoprecipitated them with anti-caspse-8 and the amounts of RIPK1, RIPK3, FADD and caspase-8 were determined in the precipitates and WCL ( d ). Data are representative of three independent experiments.
Techniques Used: Binding Assay, Immunoprecipitation
anti mouse rip3 (ProSci Incorporated)
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ProSci Incorporated manufactures this product
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Structured Review
ProSci Incorporated
anti mouse rip3
Anti Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti mouse rip3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
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Anti Mouse Rip3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti mouse rip3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
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anti mouse rip3 - by Bioz Stars,
2023-06
94/100 stars
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1) Product Images from "Membrane-bound TNF mediates microtubule-targeting chemotherapeutics-induced cancer cytolysis via juxtacrine inter-cancer-cell death signaling"
Article Title: Membrane-bound TNF mediates microtubule-targeting chemotherapeutics-induced cancer cytolysis via juxtacrine inter-cancer-cell death signaling
Journal: Cell Death and Differentiation
doi: 10.1038/s41418-019-0441-3
Figure Legend Snippet: MTAs induce MLKL phosphorylation-dependent necroptosis in L929 fibrosarcoma, both in vitro and in vivo. a Dose-dependent necroptotic cytolysis effect of MTAs on L929 cells. b A panel of 21 MTAs was tested for necroptotic effect on L929 cells. Heat map analysis of cell death index was calculated based on ATP levels. c Fluorescent microscopy of SYTOX Green-labeled necroptotic L929 cells after NCZ treatment for 24 h. Plasma membrane breakdown was traced by SYTOX Green staining. Scale bar, 400 µm. d Immunoblotting analysis of MLKL phosphorylation by Triton X-114 fractionation in whole cell lysates of NCZ-treated or PTX-treated L929 cells. T, 20 ng/ml recombinant/soluble TNF treatment. Aq, aqueous fraction; Det, detergent fraction. e Effect of Rip3 knockout on MTA-induced necroptosis in L929 cells. f Effect of RIP3 kinase activity on MTA-induced necroptosis in L929 cells. Wild-type or mutants of RIP3 were stably expressed in Rip3 KO L929 cells by pHAGE infection. WT, wild-type RIP3; K51A, kinase dead form of RIP3; S232A, auto-phosphorylation site mutant of RIP3. RIP3 re-expression was detected by immunoblotting. g In vivo response of mouse allograft of L929 cells to VCR. Athymic nude mice bearing ~300 mm 3 L929-fibrosarcoma were treated with vehicle or with 5 mg/kg Nec-1s and/or 5 mg/kg VCR. Upper: tumor growth was measured and calculated. Lower: representative image of L929 cells allografts on day 6. Vehicle, n = 5; VCR, n = 7; VCR + Nec-1s, n = 5. Scale bar, 1 cm. Graph shows mean ± SEM, p values were determined by the two-way ANOVA test; NS not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. D, DMSO; NCZ, nocodazole; VCR, vincristine; PTX, paclitaxel; DTX, docetaxel. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells ( a , b , e , and f ). Results are reported from one representative experiment. Experiments were repeated independently for four ( a , c ), three ( d – g ), or two ( b ) times
Techniques Used: In Vitro, In Vivo, Microscopy, Labeling, Staining, Western Blot, Fractionation, Recombinant, Knock-Out, Activity Assay, Stable Transfection, Infection, Mutagenesis, Expressing
Figure Legend Snippet: MTAs induce memTNF-mediated apoptosis in RIP3-deficient human carcinoma cell lines. a Immunoblotting analysis of apoptosis markers using whole cell lysates from recombinant TNF (soluble TNF, solTNF)-treated HeLa cells in the presence or absence of pan-caspase inhibitor z-VAD (Z). Cells were treated as indicated for 24 h. b Immunoblotting analysis of apoptosis markers using whole cell lysates from 1 µM MTA-treated HeLa cells in the presence or absence of 20 µM TACE inhibitor TAPI-1 (TACEi) or 20 µM pan-caspase inhibitor z-VAD (Z) for 36 h. c Immunoblotting analysis of apoptosis markers using whole cell lysates from 1 µM MTA-treated WT and TNFR1 KO HeLa cells for 36 h. d Effect of TNFR1 knockout on MTA-induced cell death in HeLa cells. Cells were treated as indicated for 48 h. e – g Immunoblotting analysis of apoptosis markers using whole cell lysates of 1 µM MTA-treated HCT116 (colon cancer, e ), MDA-MB-468 (breast cancer, f ), and BT549 (breast cancer, g ) cells for 36 h. h , i qRT-PCR analysis of JUN mRNA level ( h ) and in flow cytometric analysis of memTNF ( i ) in MTA-treated HeLa, HCT116, MDA-MB-468, and BT549 cells for 12 and 20 h respectively. D, DMSO; NCZ, nocodazole; PTX, paclitaxel; Z, z-VAD. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells ( d ). Results are reported from one representative experiment. Experiments were repeated independently three ( c , d , and h ) or two ( a , b , e – g , and i ) times
Techniques Used: Western Blot, Recombinant, Knock-Out, Quantitative RT-PCR
mouse ripk3 (ProSci Incorporated)
ProSci Incorporated is a verified supplier
ProSci Incorporated manufactures this product
94
Structured Review
ProSci Incorporated
mouse ripk3
Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
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Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse ripk3 - by Bioz Stars,
2023-06
94/100 stars
Images
1) Product Images from "Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity"
Article Title: Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.RA118.007161
Figure Legend Snippet: RIPK3 is necessary for IFN-γ–induced PS exposure in iC8KO MEFs. A, cell lysates from primary WT MEFs, primary C8KO MEFs, and iC8KO MEFs harboring the indicated expression vectors were analyzed by Western blotting with anti-RIPK3 and anti-actin antibodies. EV, an empty vector. *, nonspecific bands. B, iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and then analyzed by fluorescent microscopy. BF, Brightfield. The scale bars represent 100 μm. C, EV-expressing iC8KO (iC8KO-EV) and iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by flow cytometry. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. The quantified data of the flow cytometric analysis are presented as means ± S.D. (n ≥ 3).
Techniques Used: Expressing, Western Blot, Plasmid Preparation, Staining, Microscopy, Flow Cytometry
Figure Legend Snippet: PS is exposed in a MLKL-dependent manner in IFN-γ–treated iC8KO-RIPK3 MEFs. A and B, iC8KO-RIPK3 MEFs were infected with lentiviral vectors encoding two different shRNAs for MLKL (shMlkl#1 and shMlkl#2). The expression levels of MLKL were analyzed by qRT-PCR (A) and by Western blotting with anti-MLKL and anti-actin antibodies (B). *, nonspecific bands. C, iC8KO-RIPK3 MEFs expressing shLacZ or shMlkl#2 were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by flow cytometry. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative qRT-PCR data, Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. For qRT-PCR analysis and flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry
Figure Legend Snippet: TMEM16F is dispensable for IFN-γ–induced PS exposure. A and B, iC8KO-RIPK3 MEFs were infected with a lentiviral vector encoding shRNA for TMEM16F (sh16F). The expression of TMEM16F was quantified by qRT-PCR (A) and Western blotting with anti-TMEM16F and anti-actin antibodies (B). *, nonspecific bands. C and D, iC8KO-RIPK3 MEFs expressing shLacZ or sh16F were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by fluorescent microscopy (C) and flow cytometry (D). BF, Brightfield. The scale bar represents 100 μm. E, the quantified data of the flow cytometric analysis from D are graphically shown. Representative qRT-PCR data, Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. For qRT-PCR analysis and flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Techniques Used: Infection, Plasmid Preparation, shRNA, Expressing, Quantitative RT-PCR, Western Blot, Staining, Microscopy, Flow Cytometry
Figure Legend Snippet: The MLKL protein forms a trimer in PS-exposing MEFs treated with IFN-γ. A and B, primary WT MEFs (A) and iC8KO-RIPK3 MEFs (B) were treated with 10 ng/ml IFN-γ and 50 μm z-VAD-fmk (IZ) and with 10 ng/ml IFN-γ, respectively, for the indicated periods. Cell lysates were resolved on reducing SDS-PAGE and analyzed by Western blotting with anti-phosphorylated MLKL (pMLKL), anti-MLKL, and anti-actin antibodies. C and D, cell lysates of primary WT MEFs treated with IZ (C) or with 10 ng/ml TNFα, 1 μg/ml cycloheximide, and 50 μm z-VAD-fmk (TCZ) (D) for the indicated periods were resolved on reducing or nonreducing SDS-PAGE and analyzed by Western blotting with anti-MLKL and anti-actin antibodies. E and F, iC8KO-RIPK3 MEFs (E) or primary WT MEFs, iC8KO-RIPK3 MEFs, and iC8KO-RIPK3 MEFs expressing shMlkl#2 (F) were treated with IFN-γ for the indicated periods. Cell lysates were resolved on reducing or nonreducing SDS-PAGE and analyzed by Western blotting with anti-MLKL and anti-actin antibodies. *, nonspecific bands. Representative Western blotting data of more than three experiments are shown.
Techniques Used: SDS Page, Western Blot, Expressing
Figure Legend Snippet: Formation and shedding of membrane bubbles in IFN-γ–treated MEFs exposing PS. iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods and then directly stained with MFG-E8-GFP. The cells were then observed under fluorescent microscopy. The scale bars represent 10 μm. Representative fluorescent images of more than three experiments are shown. BF, Brightfield.
Techniques Used: Staining, Microscopy
Figure Legend Snippet: IFN-γ induces PS exposure without the induction of necroptotic cell death. A, after a 6-h treatment of iC8KO-RIPK3 MEFs with 10 ng/ml IFN-γ, IFN-γ was removed, and the cells were washed with prewarmed PBS twice and then cultured with fresh culture medium for the indicated periods. The cells at the indicated time points were stained with MFG-E8-GFP and PI and then analyzed by flow cytometry. B, the quantified data of the flow cytometric analysis from A are graphically shown. C, after a 6-h treatment of iC8KO-RIPK3 MEFs with 10 ng/ml IFN-γ, the cells were stained with MFG-E8-GFP and PI, and the MFG-E8-GFP+PI− cells were sorted and immediately analyzed by flow cytometry. Sorted cells were cultured without IFN-γ for 12 h or 2 days and analyzed by flow cytometry after staining with MFG-E8-GFP and PI. PS exposure by the sorted cells after cultivation without IFN-γ for 12 h and 2 days was again assessed by flow cytometry after a treatment with 10 ng/ml IFN-γ for 6 h. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative flow cytometric plots of more than three experiments are shown. For flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Techniques Used: Cell Culture, Staining, Flow Cytometry
mouse ripk3 (ProSci Incorporated)
ProSci Incorporated is a verified supplier
ProSci Incorporated manufactures this product
94
Structured Review
ProSci Incorporated
mouse ripk3
Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Mouse Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse ripk3/product/ProSci Incorporated
Average 94 stars, based on 1 article reviews
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mouse ripk3 - by Bioz Stars,
2023-06
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1) Product Images from "Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity"
Article Title: Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.RA118.007161
Figure Legend Snippet: RIPK3 is necessary for IFN-γ–induced PS exposure in iC8KO MEFs. A, cell lysates from primary WT MEFs, primary C8KO MEFs, and iC8KO MEFs harboring the indicated expression vectors were analyzed by Western blotting with anti-RIPK3 and anti-actin antibodies. EV, an empty vector. *, nonspecific bands. B, iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and then analyzed by fluorescent microscopy. BF, Brightfield. The scale bars represent 100 μm. C, EV-expressing iC8KO (iC8KO-EV) and iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by flow cytometry. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. The quantified data of the flow cytometric analysis are presented as means ± S.D. (n ≥ 3).
Techniques Used: Expressing, Western Blot, Plasmid Preparation, Staining, Microscopy, Flow Cytometry
Figure Legend Snippet: PS is exposed in a MLKL-dependent manner in IFN-γ–treated iC8KO-RIPK3 MEFs. A and B, iC8KO-RIPK3 MEFs were infected with lentiviral vectors encoding two different shRNAs for MLKL (shMlkl#1 and shMlkl#2). The expression levels of MLKL were analyzed by qRT-PCR (A) and by Western blotting with anti-MLKL and anti-actin antibodies (B). *, nonspecific bands. C, iC8KO-RIPK3 MEFs expressing shLacZ or shMlkl#2 were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by flow cytometry. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative qRT-PCR data, Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. For qRT-PCR analysis and flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Techniques Used: Infection, Expressing, Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry
Figure Legend Snippet: TMEM16F is dispensable for IFN-γ–induced PS exposure. A and B, iC8KO-RIPK3 MEFs were infected with a lentiviral vector encoding shRNA for TMEM16F (sh16F). The expression of TMEM16F was quantified by qRT-PCR (A) and Western blotting with anti-TMEM16F and anti-actin antibodies (B). *, nonspecific bands. C and D, iC8KO-RIPK3 MEFs expressing shLacZ or sh16F were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by fluorescent microscopy (C) and flow cytometry (D). BF, Brightfield. The scale bar represents 100 μm. E, the quantified data of the flow cytometric analysis from D are graphically shown. Representative qRT-PCR data, Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. For qRT-PCR analysis and flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Techniques Used: Infection, Plasmid Preparation, shRNA, Expressing, Quantitative RT-PCR, Western Blot, Staining, Microscopy, Flow Cytometry
Figure Legend Snippet: The MLKL protein forms a trimer in PS-exposing MEFs treated with IFN-γ. A and B, primary WT MEFs (A) and iC8KO-RIPK3 MEFs (B) were treated with 10 ng/ml IFN-γ and 50 μm z-VAD-fmk (IZ) and with 10 ng/ml IFN-γ, respectively, for the indicated periods. Cell lysates were resolved on reducing SDS-PAGE and analyzed by Western blotting with anti-phosphorylated MLKL (pMLKL), anti-MLKL, and anti-actin antibodies. C and D, cell lysates of primary WT MEFs treated with IZ (C) or with 10 ng/ml TNFα, 1 μg/ml cycloheximide, and 50 μm z-VAD-fmk (TCZ) (D) for the indicated periods were resolved on reducing or nonreducing SDS-PAGE and analyzed by Western blotting with anti-MLKL and anti-actin antibodies. E and F, iC8KO-RIPK3 MEFs (E) or primary WT MEFs, iC8KO-RIPK3 MEFs, and iC8KO-RIPK3 MEFs expressing shMlkl#2 (F) were treated with IFN-γ for the indicated periods. Cell lysates were resolved on reducing or nonreducing SDS-PAGE and analyzed by Western blotting with anti-MLKL and anti-actin antibodies. *, nonspecific bands. Representative Western blotting data of more than three experiments are shown.
Techniques Used: SDS Page, Western Blot, Expressing
Figure Legend Snippet: Formation and shedding of membrane bubbles in IFN-γ–treated MEFs exposing PS. iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods and then directly stained with MFG-E8-GFP. The cells were then observed under fluorescent microscopy. The scale bars represent 10 μm. Representative fluorescent images of more than three experiments are shown. BF, Brightfield.
Techniques Used: Staining, Microscopy
Figure Legend Snippet: IFN-γ induces PS exposure without the induction of necroptotic cell death. A, after a 6-h treatment of iC8KO-RIPK3 MEFs with 10 ng/ml IFN-γ, IFN-γ was removed, and the cells were washed with prewarmed PBS twice and then cultured with fresh culture medium for the indicated periods. The cells at the indicated time points were stained with MFG-E8-GFP and PI and then analyzed by flow cytometry. B, the quantified data of the flow cytometric analysis from A are graphically shown. C, after a 6-h treatment of iC8KO-RIPK3 MEFs with 10 ng/ml IFN-γ, the cells were stained with MFG-E8-GFP and PI, and the MFG-E8-GFP+PI− cells were sorted and immediately analyzed by flow cytometry. Sorted cells were cultured without IFN-γ for 12 h or 2 days and analyzed by flow cytometry after staining with MFG-E8-GFP and PI. PS exposure by the sorted cells after cultivation without IFN-γ for 12 h and 2 days was again assessed by flow cytometry after a treatment with 10 ng/ml IFN-γ for 6 h. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative flow cytometric plots of more than three experiments are shown. For flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Techniques Used: Cell Culture, Staining, Flow Cytometry