The collected tissue was first well-ground using a homogeniser, lysed by adding RIPA lysis (P0013B, Beyotime, China) solution for 20 min, and then centrifuged at high speed (13,500 rpm for 15 min). Cell lysis was also prepared using the RIPA method. The protein samples were separated on SDS-PAGE (PG112, Epizyme Biotech, Shanghai, China) and transferred to PVDF membranes (ISEQ00010, Millipore, USA). The resulting blots were incubated with primary antibides against hook microtubule tethering protein 1 (HOOK1) (1:750, K003840P, Solarbio, Beijing, China), CUL4A (1:1000, K008800P, Solarbio, Beijing, China), MLKL (1:1000, Cat.#: 380559, Zen Biotechnology Co., Ltd, Chengdu, China), Phospho-MLKL (Ser358) (1:1250, Cat# 382136, Zen Biotechnology Co., Ltd, Chengdu, China), RIPK3 (1:1000, Cat# AF4808, RRID: AB_2844793 , Affinity Biosciences, China), Phospho-RIPK3 (3:5000, Cat# 93654, RRID: AB_2800206 , Cell Signaling Technology, China), RIPK1 (1:1000, Cat# 3493, RRID: AB_2305314 , Cell Signaling Technology, China), GAPDH (1:2000, Cat# 60004-1-Ig, RRID: AB_2107436 , Proteintech, Wuhan, China), and the secondary antibody (1:2000, Cat.# A212020, Abbkine, China; 1:2000, SA00001-1, Proteintech, China). For details, please refer to the previous publication.56 (link)
Phospho ripk3
Manufactured by Cell Signaling Technology
Sourced in China, United States
Phospho-RIPK3 is a lab equipment product from Cell Signaling Technology. It is an antibody that specifically detects the phosphorylated form of the Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab183218, by Abcam (2 mentions)
Ab40676, by Abcam (2 mentions)
Ab168352, by Abcam (2 mentions)
Cd163, by BioLegend (2 mentions)
Phospho nf κb, by Cell Signaling Technology (2 mentions)
Ripk3, by Cell Signaling Technology (2 mentions)
Ab178945, by Wuhan Servicebio Technology (2 mentions)
Hrp conjugated anti rabbit and anti mouse secondary antibodies, by Boster Bio (2 mentions)
Nf κb, by Proteintech (2 mentions)
Phospho tbk1 nak ser172, by Cell Signaling Technology (2 mentions)
Gapdh, by Wuhan Servicebio Technology (2 mentions)
M csf, by BioLegend (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
F4 80, by BioLegend (2 mentions)
Iseq00010, by Merck Group (1 mentions)
Ripk3, by Affinity Biosciences (1 mentions)
Ripk1, by Cell Signaling Technology (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Phospho mlkl, by Cell Signaling Technology (1 mentions)
4 protocols using phospho ripk3
1
Protein Extraction and Western Blot Analysis
2
Necroptosis Pathway Protein Analysis
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Caspase-3, Caspase-8, RIPK1, RIPK3 (rabbit antibody), MLKL, OGT and OGA antibodies were purchased from Proteintech (Wuhan, China). Phospho-RIPK1, phospho-RIPK3, phospho-MLKL and secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA). MPO antibody was provided by ABclonal Technology (Wuhan, China). CD68 antibody was from Abcam (Cambridge, MA). O-GlcNAc antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Another RIPK3 (mouse) antibody was purchased from Novus Biologicals (Littleton, CO, USA). 2,4,6-trinitrobenzene sulfonic acid (TNBS) was from Thermo Fisher Scientific (CA, USA). Protein A/G magnetic beads were purchased form Bimake (Shanghai, China). The TUNEL staining and immunohistochemistry kits were purchased from Wuhan Gugeshengwu Technology Co.,Ltd. (Wuhan, China). All other regular reagents were from Wuhan Gugeshengwu Technology Co.,Ltd. unless otherwise specified.
3
Immune Signaling Pathway Profiling
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We used antibodies against TBK1 (Abcam, ab40676, 1:1000), phospho-TBK1/NAK (Ser172) (Cell Signaling, #5483, 1:1000), RIPK3 (Cell Signaling, #95702, 1:1000), phospho-RIPK3 (Cell Signaling, #91702, 1:1000), NF-κB (Proteintech, 10745-1-AP, 1:1000), phospho-NF-κB (Cell Signaling, #3033, 1:1000), GLUD1 (Abcam, ab168352, 1:1000), TNF-α (Abcam, ab183218, 1:1000), iNOS (Abcam, ab178945, 1:1000), GAPDH (Servicebio, GB12002, 1:1000). CD86 (Biolegend,105007), CD163 (Biolegend, 333606), and F4/80 (Biolegend, 123109). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Boster Biological Technology (Wuhan, China). LPS was purchased from Sigma (L2880). Neutral liposomes and control liposomes (F70101C-NC-2) were purchased from FormuMax. M-CSF (576402) was obtained from Biolegend.
4
Immune Signaling Pathway Profiling
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We used antibodies against TBK1 (Abcam, ab40676, 1:1000), phospho-TBK1/NAK (Ser172) (Cell Signaling, #5483, 1:1000), RIPK3 (Cell Signaling, #95702, 1:1000), phospho-RIPK3 (Cell Signaling, #91702, 1:1000), NF-κB (Proteintech, 10745-1-AP, 1:1000), phospho-NF-κB (Cell Signaling, #3033, 1:1000), GLUD1 (Abcam, ab168352, 1:1000), TNF-α (Abcam, ab183218, 1:1000), iNOS (Abcam, ab178945, 1:1000), GAPDH (Servicebio, GB12002, 1:1000). CD86 (Biolegend,105007), CD163 (Biolegend, 333606), and F4/80 (Biolegend, 123109). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Boster Biological Technology (Wuhan, China). LPS was purchased from Sigma (L2880). Neutral liposomes and control liposomes (F70101C-NC-2) were purchased from FormuMax. M-CSF (576402) was obtained from Biolegend.
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