Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Necrotic-like morphology was observed in PANC1 cells following treatment with CCT137690 (10 μM) for 24 hours. (B) Western blot analysis of indicated proteins in PANC1 and PANC2.03 cells following treatment with CCT137690 (2.5–10 μM) for 24 hours (n=3, *p < 0.05 versus untreated group). (C, D) PANC1 and PANC2.03 cells were treated with CCT137690 (C) or staurosporine (D) or erastin (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with CCT137690 (10 μM) for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F–I) Knockdown of RIPK1, RIPK3, and MLKL by shRNAs inhibited CCT137690 (10 μM, 24 hours)-induced cell death and HMGB1 and ATP release (n=3, *p < 0.05 versus control shRNA group).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Knockdown, Control, shRNA

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PDAC cells (n=3, *p < 0.05 versus control shRNA group). (C) Immunoprecipitation (IP) analysis of the levels of RIPK3 binding to RIPK1 and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (D) IP analysis of the levels of RIPK3 binding to RIPK1 and MLKL in control and stable AURKA-knockdown PANC1 and PANC2.03 cells. (E) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (F) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in HEK293 cells after expressed AURKA wild type and D274A mutant. (G) PANC1 cells were treated with CCT137690 (10 μM) or TNF (50 ng/ml) in the absence or presence of anti-TNFR1 antibody (1 mg/ml) for 24 hours and cell death was analyzed (n=3, *p < 0.05).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Control, Knockdown, shRNA, Immunoprecipitation, Binding Assay, Mutagenesis

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PANC1 and PANC2.03 cells (n=3, *p < 0.05 versus control shRNA group). (C, D) PANC1 and PANC2.03 cells were treated with XXVI (C) or AR-A014418 (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with XXVI or AR-A014418 for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F) Knockdown of RIPK3 and MLKL, but not RIPK1, inhibited XXVI- or AR-A014418-induced cell death (n=3, *p < 0.05 versus control shRNA group). (G–I) HEK293 cells were expressed with GSK3β WT and S9A mutant and then treated with CCT137690 (10 μM) for 24 hours. Protein level (G), cell viability (H), caspase-3 activity (H), and complex formation (I) were assayed.

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Control, Knockdown, shRNA, Mutagenesis, Activity Assay

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: ( A ) Histology of untreated cultures (UT) show (a) low-grade RCC with small round tumor cells and a clear cytoplasm surrounded by a distinct cell membrane and round uniform nuclei with inconspicuous or absent nucleoli. (b) R1TNF- and (c) wtTNF-treated cultures show elevated level of death in mTECs compared with (d) R2TNF-treated cultures. ( B ) Morphological features of necrosis are evident in R1TNF-treated cultures such as large/small cytoplasmic vacuoles (x), condensation of the chromatin into small, irregular, circumscribed patches (white arrow), increasing translucent cytoplasm, swollen organelles (orange arrow) and disruption of the plasma membrane (black arrows). ( C ) Expression of RIPK1 and RIPK3 in tissue biopsies comprising RCC and NK; NK show a rare signal for RIPK1 and RIPK3 in MNCs within glomeruli (arrows) and with interstitium (small arrows). In contrast, RCC grade 3 shows marked signal in mTECs (arrows), MNCs (small arrows) and in some VECs (arrowheads). ( D and E ) Representative immunoblot of samples from nine patients with similar results and bar graph of relative RIPK1 and RIPK3 protein levels in grades 3/4 RCC (G3/G4) and non-tumor kidney (NK). Bars=mean+S.E.M.; * P <0.05 versus NK; paired Student's t -test. Glom-glomeruli; MNCs-mononuclear cells; TECs-tubular epithelial cells

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Membrane, Disruption, Clinical Proteomics, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: Representative confocal images and light micrographs of the effect of R1TNF on protein and mRNA expression for RIPK1 and RIPK3 in organ cultures of RCC grade 1 and adjacent non-tumor kidney (NK). ( a and b ) Untreated (UT) cultures from RCC show a rare signal for RIPK1 and RIPK3 protein and mRNA. In contrast, R1TNF-treated cultures show a marked signal of both proteins mainly confined to mTECs (arrows), with RIPK3 also present in nuclei (white shaded arrow). Similarly, UT cultures of NK show a rare signal for both proteins; increased expression of both protein and mRNA is detected in R1TNF-treated cultures mainly confined to normal TECs (white arrows), peritubular capillaries (white arrowheads) and in infiltrating mononuclear cells (black arrowhead) but not in glomeruli (Glom). ( c ) Representative mean fluorescence intensity (MFI) for RIPK1 and RIPK3 expression in RCC and NK organ cultures. ** P <0.01 versus UT, * P <0.05 versus UT, ± P <0.05 versus R1TNF; Bars=mean±S.E.M.; n =3 independent experiments from six separate organ culture experiments with similar results. Nuclei stained with Hoechst 33342. Confocal images: × 40 and × 63 original magnifications; photomicrographs: × 400 magnification

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Expressing, Fluorescence, Organ Culture, Staining

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: PLA of organ culture of RCC grade 1. ( a ) In comparison with untreated controls (UT), R1TNF induced a strong interaction of RIPK1-RIPK3 and RIPK3-pMLKL Ser358 appearing as strong red fluorescence spots mainly within the cytoplasm of mTECs. Each individual interacting protein pair observed as a red spot by confocal microscopy is expressed as the number of signals/cell (PLA spots/cell). ( b and c ) Quantification of PLA spots in TECs in the two study groups; RCC (RCCoC) and normal kidney (NKoC) show a statistically significant difference, more pronounced in RCCoC. ** P <0.01 versus UT, * P <0.05 versus UT, ± P <0.05 versus R1TNF (NKoC). ( d and e ) Representative confocal images of pMLKL Ser358 or pDrp1 Ser637 and TUNEL in organ cultures of RCC grade 1. Compared with UT cultures, R1TNF induced an increase in the level of TUNEL+ mTECs ( green ) associated with pMLKL Ser358 and pDrp1 Ser616 ( red ) expression (arrows). ( f and g ) Quantification of TUNEL+ mTECs/pMLKL Ser358+ and TUNEL+ mTECs/pDrp1 Ser616+ shows statistical significant differences between cultures. *** P <0.0001 versus UT, ** P <0.001 versus UT, * P <0.01 versus UT, ± P <0.05 versus R1TNF (NKoC). ( h ) Combined immunofluorescence of R1TNF-treated cultures shows co-localization of pMLKL Ser358 and pDrp1 Ser616 in mTECs (arrow). ( i ) Immunogold electron microscopy demonstrate close proximity of gold particles for pMLKL Ser358 (5 nm) and pDrp1 Ser616 (15 nm) in mitochondria (m) ( inset zoomed × 2.5 ). Bars=mean±S.E.M.; images are representative of n =3 independent experiments from six separate organ culture experiments with similar results

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Organ Culture, Comparison, Fluorescence, Confocal Microscopy, TUNEL Assay, Expressing, Immunofluorescence, Electron Microscopy

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: Expression of necrosomal signaling components regulated by wtTNF, R1TNF and R2TNF in malignant tubular epithelial cells in human RCC grade 1 organ cultures

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: Expressing, Immunostaining

Journal: Cell Death & Disease

Article Title: Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis

doi: 10.1038/cddis.2016.184

Figure Lengend Snippet: ( a ) Effect of necrosulfonamide (NSA) and mdivi-1 (m) in grade 1 RCC organ cultures treated with R1TNF. R1TNF alone without NSA (no NSA) induced increased levels of TUNEL+ mTECs, which were significantly reduced with 50 μ M NSA, and to a lesser extent, with 10 μ M or 20 μ M NSA (arrows). Cultures pretreated with 5 μ M NSA showed comparable levels of TUNEL+ mTECs as untreated cultures (UT). ( b ) The percentage of TUNEL+ mTECs and ( c ) pMLKL Ser358 expression presented as mean fluorescent intensity (MFI) in similar cultures. *** P <0.0001 versus UT, ** P <0.001 versus R1TNF; * P <0.05 versus R1TNF; ¥ P <0.05 versus R1TNF (+20 or 10 μ M); ± P <0.001 versus R1TNF (+20 or 10 μ M); ns, not significant. ( d ) In comparison with UT, which show a rare TUNEL+ mTECs, R1TNF alone (without m) induced increased levels of TUNEL+ mTECs, significantly reduced by m (10 μ M) with no effect by zVAD.fmk (m+z) but a marked reduction by nec-1 (m+n) comparable with cultures pretreated with a combination of zVAD.fmk and nec-1 (m+n+z). ( e ) The percentage of TUNEL+ mTECs and ( f and g ) the mean fluorescence intensity (MFI) for pDrp1 Ser616 and pDrp1 Ser637 in similar cultures. *** P <0.0001 versus UT ( e ), ** P <0.01 versus UT ( f ), * P <0.05 versus R1TNF, ± P <0.05 versus R1TNF+m. ( g ) * P <0.05 versus UT, ± P <0.5 versus R1TNF, ¥ P <0.05 versus R1TNF+m; ns, not significant. Bars=mean±S.E.M.; n =3 independent experiments from six separate organ culture experiments with similar results. ( h ) Schematic diagram of the consequences of R1TNF-mediated necroptosis in mTEC in RCC; ligation of TNFR1 results in the recruitment of RIPK1, facilitating its interaction with RIPK3, which in turn recruits and phosphorylates MLKL at Ser358 and Drp1 at Ser616 thus causing their co-localization with the mitochondria. A separate process causes a reduction in pDrp1 at ser637. Nec-1 inhibits RIPK1, and NSA inhibits MLKL and mdivi-1 inhibits Drp1 inhibiting cell death

Article Snippet: Blocking peptides for RIPK1 (cat~NBP1-77077PEP) are from Novus Biologicals and for RIPK3 (~ab178834) and pMLKL Ser358 (ab206929) are from Abcam. zVAD.fmk (pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; cat~G7231) is from Promega, Madison, WI, USA; PDTC, 3,3′-diaminobenzidine substrate (DAB), Necrostatin-1 (Nec-1, cat~N9037) and mdivi-1 (3-(2, 4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone) are from Sigma-Aldrich (Gillingham, UK).

Techniques: TUNEL Assay, Expressing, Comparison, Fluorescence, Organ Culture, Ligation

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) To gauge immunohistochemistry performance, immunosignals from wild-type (WT) versus knockout (KO) tissue were deconvoluted, (i) pixel intensities plotted, (ii) ratioed to yield a signal-to-noise (S/N) histogram, and then (iii) integrated. ( B ) Heatmap shows relative integrated S/N values from seven automated immunohistochemistry protocols across seven tissues. Column headers indicate the antibody target clone name. Data were representative of n ≥ 3 for each target and tissue. ( C ) Heatmap depicts relative protein abundance values as SILAC (stable isotope labeling by amino acids in cell culture) ratios measured by (Geiger et al, ); the lowest value was assigned as 0.1 because 0 is below the detection limit. ( D ) Immunosignals of Caspase-8, RIPK1, RIPK3, and MLKL in wild-type versus the appropriate knockout (KO) tissue from Mlkl −/− or Casp8 − / − Ripk3 − / − or Casp8 −/ − Ripk1 −/ − Ripk3 − /− . Data were representative of n ≥ 3 for each target and tissue. Scale bars are 500 μm. Related to Appendix Fig. . .

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Immunohistochemistry, Knock-Out, Labeling, Cell Culture

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) Immunosignals of Caspase-8, RIPK1, and RIPK3 from wild-type mouse ileum (i), colon (ii), liver (iii), and spleen (iv). The crypt base (crypt), villi/crypt tip (tip), central vein (CV), portal vein (PV), bile duct (BD), central artery (CA), white pulp (WP), marginal zone (MZ), and red pulp (RP) are annotated. Inset of immunostaining in the ileum shows lower RIPK3 expression in Paneth cells (open arrowhead) relative to neighboring cells. Arrow shows pericentral hepatocytes that express higher levels of Caspase-8. Closed arrowheads show Caspase-8 + RIPK1 + RIPK3 + Kupffer cells. Scale bars are 50 μm, except for the 10 μm scale bar in the inset. Data were representative of n ≥ 3 for each target and tissue. ( B ) Relative expression levels of Caspase-8, RIPK1, and RIPK3 (and splenic MLKL; Fig. ) along the indicated tissue axes. Red datapoints indicate immunosignal intensities, and the overlaid dark blue line indicating the LOWESS best-fit along N = 20 axes per tissue. Best-fit curves are superimposed in the left-most column. The dashed line indicates the boundary between the splenic white pulp and the marginal zone. Data were representative of n > 3 mice per target per tissue. ( C ) Scatterplots where each dot represents a different cell ontology from the Tabula Muris dataset (Tabula Muris et al, ). The percent of cells within each ontology that expressed Mki67 , Ripk1 , or Ripk3 was plotted against that of Top2a . Pearson correlation coefficient values are shown. Related to Fig. . .

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Immunostaining, Expressing

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) Heatmap of cell ontologies from the Tabula Muris dataset (Tabula Muris et al, ). Left-most column depicts the tissue origin of each cell ontology. Other columns indicate the percent of cells within each ontology that expressed Top2a, Mki67 , Casp8 , Ripk1, Ripk3 , or Mlkl . Legend shows the color-to-tissue and the color-to-frequency scales. Cell ontologies of interest are annotated. ( B ) Micrograph of MLKL immunosignals from the wild-type mouse spleen. The white pulp (WP), marginal zone (MZ), and red pulp (RP) are annotated. Scale bar is 50 μm. Scatterplot shows relative expression levels of MLKL along the white pulp-to-red pulp axis. Red datapoints show immunosignal intensities and the overlaid dark blue line indicates the LOWESS best-fit along N = 20 axes from n = 1 mouse. Dashed line indicates the boundary between splenic white pulp and marginal zone. Data were representative of n > 3 mice. ( C , D ) Spatial transcriptomic data from (Moor et al, )) and (Ben-Moshe et al, ) showing the relative expression levels (arbitrary units; A.U.) of Caspase-8, RIPK1, RIPK3, or MLKL along the ileal crypt-to-villus axis ( C ) or the hepatic central vein-to-portal vein axis ( D ). ( E – G ) Spatial transcriptomic data on mouse spleen 12 days after Plasmodium berghei- infection. Panel ( E ) shows a uniform manifold approximation and projection (UMAP) of cell populations distinguished by unsupervised leiden clustering. Legend shows the color assigned to each population. Panel ( F ) shows the location of each cell cluster. Scale bar is 500 μm. Panel ( G ) shows the normalized expression for each gene product. Expression values for Casp8 , Ripk1 , Ripk3 , and Mlkl were summated to provide an index of “cluster pathway expression”, which was averaged to provide an index of “zone pathway expression”. Data were from n = 1 mouse.

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Expressing, Infection

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Core temperatures of vehicle- and TNF-injected mice ( n = 3 mice per group). ( C ) Immunosignals for cleaved Caspase-3 (cl. C3), Caspase-8, RIPK1, or RIPK3 from the spleen of vehicle- or TNF-injected mice. Insets show unidentified RIPK1 high cells that associate with apoptotic bodies in the splenic white pulp. In panels ( C , E , G , I ), the scale bars in zoomed-out images correspond to 100 μm, and the scale bars in zoomed-in images correspond to 10 μm. ( D ) Graph of white pulp area occupied by cleaved Caspase-3 + material in vehicle- and TNF-treated mice. Each red datapoint represents one white pulp lobule ( N = 20 lobules/mouse). Blue datapoints indicate the median value per mouse ( n = 3 mice/treatment). Black bars represent the mean value per group. * p < 0.05 by unpaired two-tailed t -test. ( E ) RIPK3 immunosignals in the colon of vehicle- or TNF-treated mice. ( F ) Best-fit curves of RIPK3 immunosignals along the crypt-to-tip axis from N = 10 axes per mouse ( n = 3 mice/group). *** p < 0.001 by multiple unpaired two-tailed t -test. ( G ) RIPK3 (pink) and smooth muscle actin (brown) immunosignals in intestinal submucosa of vehicle- or TNF-treated mice. Insets show vessel cross-sections. Arrowheads show RIPK3 + endothelial cells (endo). ( H ) Plot of RIPK3 signals per vessel. Each red datapoint represents one vessel ( N = 50 vessels/mouse). Blue datapoints indicate the median value per mouse ( n = 3 mice/treatment). Black bars represent the mean value per group. * p < 0.05 by unpaired two-tailed t -test. ( I ) RIPK3 immunosignals in the liver of vehicle- or TNF-treated mice. Central vein (CV), portal vein (PV) and Kupffer cell (Kpf). ( J ) Plot of RIPK3 signals per hepatocyte or Kupffer cell. Each transparent datapoint represents one cell ( N = 90 cells/mouse). Opaque datapoints indicate the median value per mouse ( n = 3 mice/treatment). Black bars represent the mean value per group. * p < 0.05 and ** p < 0.01 by unpaired two-tailed t -test. ( K ) Core temperatures of vehicle-, TNF- and Nec1s+TNF-injected wild-type mice ( n = 4–5 mice/treatment; one dot/mouse/time). Line indicates mean. X indicates a euthanized mouse due to its body temperature being <30 °C. ( L ) RIPK3 levels in serum from the mice in panel ( K ) or from untreated Ripk3 -/- mice. Data expressed as arbitrary optical density units (A.U.). One dot per mouse. Mean ± SEM is shown. * p < 0.05, ** p < 0.01 by one-way ANOVA with Tukey’s post hoc correction. .

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Injection, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Bulk RNA sequencing was performed on indicated tissues. Heatmap depicts the log-fold-change in gene expression for antibiotic- versus water-treated mice. Each row represents a different mouse. The legend shows the color-to-value scale. ( C ) Immunoblots for the indicated proteins in the ileum and spleen of water- versus antibiotic-treated mice. Arrowheads indicate full-length proteins of interest. Coomassie staining of total protein content was used as a loading control. Data were representative of n = 7 mice per tissue per group. ( D ) Caspase-8, RIPK3, and RIPK3 immunosignals in the ileum of water- or antibiotic-treated mice. Arrowheads to cytosolic accumulations of RIPK1 and RIPK3 in epithelial cells at villi tips. Scale bars in lower magnification micrographs are 100 μm. Scale bars in insets are 10 μm. Data were representative of n = 7 mice per group. Related to Appendix Fig. . .

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: RNA Sequencing Assay, Expressing, Western Blot, Staining, Control

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Ki67, Caspase-8, RIPK1, RIPK3, and MLKL immunosignals from adjacent sections of the naïve or NP-KLH-immunized mouse spleen. Arrowheads show a Ki67 + germinal center that co-stains for RIPK3, but not other members of the pathway. Representative of n > 3 mice per group. Scale bars in lower magnification micrographs are 500 μm. Scale bars in insets are 100 μm. Data were representative of n > 3 mice per group. ( C – J ) Ripk3 -/- or Ripk3 +/+ mice were immunized with NP-KLH and circulating NP-specific IgG 1 ( C ), circulating NP-specific IgM ( D ), splenic mature B cells ( E , F ), splenic NP-specific plasma cells ( G ), circulating low affinity NP-specific IgG 1 antibody ( H ), circulating high affinity NP-specific IgG 1 ( I ), and the ratio between circulating low-and-high affinity NP-specific antibody ( J ) were measured at the indicated day after immunization. Bars on graphs in ( C – J ) represent mean ± SEM. Each datapoint represents one mouse. ns non-significant by two-sided t -test with Welch’s correction.

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) Immunosignals of cleaved Caspase-3, Caspase-8, RIPK1, RIPK3, and MLKL in wild-type versus MLKL -/- or RIPK1 -/- , or CASP8 -/- CASP10 -/- MLKL -/- HT29 cells. Arrowheads indicate Caspase-8 + , RIPK1 + , RIPK3 + , and MLKL + puncta that are presumed to be necrosomes. Data were representative of n ≥ 2 for each protein and treatment. Scale bars in lower magnification micrographs are 10 μm. Scale bars in insets are 2 μm. ( B ) The percent of cells per treatment group that contain cytosolic necrosome-like puncta immunostained by the stipulated antibody. N = 1051–5630 cells were analysed per condition per stain. Data representative of n = 2 experiments. ( C ) The number of puncta per cell. N = 1000 cells per treatment group were analysed. Each datapoint represents one cell. The black bar indicates the mean value. **** p < 0.0001 by one-way ANOVA with Krukal–Wallis post hoc correction. Data representative of n = 2 experiments. Related to Appendix Figs. , and . .

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: ( A ) Approach used to insert the human RIPK3 coding sequence (CDS) into the mouse Ripk3 locus. ( B ) Immunoblot of spleen homogenates from Ripk3 +/+ , Ripk3 -/- , human RIPK3 ( RIPK3 KI/KI ) or hemizygous human RIPK3 ( Ripk3 + RIPK3 KI ) mice. GAPDH immunoblots are shown as loading controls. Each lane represents a different mouse. ( C ) Immunosignals produced by the anti-human RIPK3 (clone 37A7F) or anti-mouse RIPK3 (clone 8G7) antibodies on spleen sections from RIPK3 KI/KI , Ripk3 +/+ or Casp8 -/- Ripk1 -/- Ripk3 -/- . Data were representative of n ≥ 3 for each target and tissue. Scale bars are 500 μm.

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Sequencing, Western Blot, Produced

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: Immunohistochemistry for cleaved Caspase-3, Caspase-8 (clone B.925.8), RIPK1, RIPK3, and MLKL (clone EPR171514) on intestinal biopsies from the stipulated patients. Four representative micrographs per biopsy are shown (i-v). Open arrowheads indicate instances of epithelial apoptosis. Closed arrowheads indicate instances of epithelial Caspase-8 clustering. Scale bars are 10 μm. The location for each micrograph within the biopsy is indicated in Appendix Fig. .

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: A range of working dilutions were trialed for the following antibodies, although no conditions could be optimized for specificity and intensity: rabbit anti-phospho-RIPK1 (clone D813A; RRID:AB_2799268; Cell Signaling Technology Cat#44590 S); mouse anti-RIPK1 (clone 38/RIP; RRID:AB_397831; 0.25 g/L BD Biosciences Cat#610459); mouse anti-RIPK1 (clone 334640; RRID:AB_2253447; 0.5 g/L; R&D Systems Cat#MAB3585); rabbit anti-RIPK3 (clone 18H1L23; RRID: AB_2866471; 0.5 g/L; Thermo Fisher Scientific Cat#703750); rabbit anti-phospho-RIPK3 (clone D6W2T; RRID:AB_2800206; Cell Signaling Technology Cat#93654); rabbit anti-phospho-MLKL (clone D6E3G; RRID:AB_2799112; Cell Signaling Technology Cat# 37333); rabbit anti-phospho-MLKL (clone EPR9514; RRID:AB_2619685; Abcam Cat#ab187091; (Wang et al, )); mouse anti-MLKL (clone 3D4C6; RRID:AB_2882029; 1.957 g/L; ProteinTech Cat#66675-1-IG); rat anti-MLKL (clone 3H1; RRID:AB_2820284; 2 g/L produced in-house (Murphy et al, ) and available from Millipore Cat# MABC604); rabbit anti-mouse MLKL (clone D6W1K; RRID:AB_2799118; Cell Signaling Technology Cat#37705); rabbit anti-MLKL (clone 2B9; RRID:AB_2717284; 1 g/L; Thermo Fisher Scientific Cat#MA5-24846); mouse anti-Caspase-10 (clone 4C1; RRID:AB_590721; 1 g/L; MBL International Cat# M059-3); mouse anti-Caspase-8 (clone 5D3; RRID:AB_590761; 1 g/L; MBL International Cat#M058-3); rat anti-human RIPK3 (clone 1H2; RRID:AB_2940816; 2 g/L produced in-house (Petrie et al, ) and available from Millipore Cat# MABC1640); rabbit anti-human RIPK3 (clone E1Z1D; RRID:AB_2687467; Cell Signaling Technology Cat# 13526); rat anti-human MLKL (clone 7G2; RRID:AB_2940818; 2 g/L produced in-house (Samson et al, ) and available from Millipore Cat# MABC1636).

Techniques: Derivative Assay, Polymer, Plasmid Preparation, Blocking Assay, Membrane, Staining, RNA HS Assay, dsDNA Assay, Protease Inhibitor, Software, Imaging, Enzyme-linked Immunosorbent Assay