Whole embryos were snap-frozen and homogenised in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40 and 1xEDTA-free proteinase inhibitor cocktail (Roche, 5056489001) or RIPA buffer with 6M Urea for the experiment in Extended data Fig 7h . Alternatively, cells were washed twice with ice-cold PBS prior to lysis in lysis buffer. Protein concentration of lysates was determined using BCA protein assay (Thermo Scientific). Lysates were subsequently denatured in reducing sample buffer at 95°C for 10 min before separation by SDS-PAGE (NuPAGE) and subsequent analysis by Western blotting using antibodies against HOIL-130 (link), HOIP (custom-made, Thermo Fisher Scientific), SHARPIN (ProteinTech, 14626-1-AP), TNFR1 (Abcam, ab19139), Actin (Sigma, A1978), pIκBα (Cell Signaling, 9246), IκBα (Cell Signaling, 9242), cleaved caspase-8 (Cell Signaling, 9429), linear ubiquitin (Merck Millipore, MABS199), RIPK1 (BD, 610459), RIPK3 (Enzo, ADI-905-242-100), FADD (Assay Design, AAM-121), MLKL (Millipore, MABC604), phospho-MLKL (Abcam, ab196436) and Tubulin (Sigma, T9026).
Mabc604
Manufactured by Abcam
MABC604 is a lab equipment product designed for use in scientific research. It serves a core function within the research process, but a detailed description while maintaining an unbiased and factual approach is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
P iκbα, by Cell Signaling Technology (2 mentions)
Proteinase inhibitor cocktail, by Roche (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Mabs199, by BD (2 mentions)
Cleaved caspase 8, by Cell Signaling Technology (2 mentions)
Lsm 800, by Zeiss (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
Freezing microtome, by Leica (1 mentions)
Anti mlkl, by Merck Group (1 mentions)
Anti myd88, by R&D Systems (1 mentions)
Anti rip3, by Santa Cruz Biotechnology (1 mentions)
3 protocols using mabc604
1
Protein Extraction and Western Blotting
2
Immunostaining of Necroptosis Pathway
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The discs were fixed by 4% paraformaldehyde (PFA) for 24h at 4°C, followed by dehydration in 25% sucrose solution for 48 h. The tissues were then implanted into optimal cutting temperature compound (Sakura), and serial 12-μm-thick sections were cut on freezing microtome (Leica). All sections were placed on slides and stored at -20°C for immunostaining.
After blocking with 0.01M PBS solution containing 3% BSA and 0.3% Triton X-100 for 30 min, the sections were incubated with primary antibodies in a humid chamber at 4°C overnight. Anti-RIP3(1:300, Santa Cruz Biotechnology, sc-374639), anti-MLKL(1:200, Milipore, MABC 604), anti- p-MLKL(1:200, Abcam, ab187091), and anti-MyD88(1:400, R&D, MAB2928) antibodies were used in this study. The slides were then washed by 0.01M PBS three times, and incubated with corresponding secondary antibodies. The nuclei were stained with Hoechst 33342. The images were photographed by confocal microscope (LSM 800, Zeiss).
After blocking with 0.01M PBS solution containing 3% BSA and 0.3% Triton X-100 for 30 min, the sections were incubated with primary antibodies in a humid chamber at 4°C overnight. Anti-RIP3(1:300, Santa Cruz Biotechnology, sc-374639), anti-MLKL(1:200, Milipore, MABC 604), anti- p-MLKL(1:200, Abcam, ab187091), and anti-MyD88(1:400, R&D, MAB2928) antibodies were used in this study. The slides were then washed by 0.01M PBS three times, and incubated with corresponding secondary antibodies. The nuclei were stained with Hoechst 33342. The images were photographed by confocal microscope (LSM 800, Zeiss).
3
Protein Extraction and Western Blotting
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