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a – d , HT - 29 cells with the indicated knockouts ( a ) or knockdowns ( b – d ) were treated with TSE (30 ng ml −1 hTNF, 0.2 µM SM-164, 5 µM emricasan) or TRAIL + SE (50 ng ml −1 , 0.2 µM SM-164, 5 µM emricasan) for 8 h, and cell death or cell viability was quantified using the indicated assays ( P = 1.39 × 10 −13 ( a ), P < 1 × 10 −15 ( b ), P < 1 × 10 −15 ( c ) and P = 6.82 × 10 −12 , P = 1.23 × 10 −11 ( d )). Cell lysates were analysed using reducing or non-reducing <t>gel</t> <t>electrophoresis</t> with the indicated antibodies. e , HT-29 cells with indicated stable short hairpin RNA (shRNA)-mediated knockdowns were treated with TSE for the indicated times, and cell death was assessed using CellToxGreen and time-lapse fluorescence confocal microscopy. f , As in e , except cell morphology was visualized by scanning electron microscopy. g , As in e , except the release of HMGB1 was analysed by immunoblotting. Data are plotted as the mean ± s.d., representative of n = 4 ( c ), n = 9 ( a , d ) or n = 12 ( b ); three independent experiments. Statistical analyses were performed using two-tailed t -tests and two-way analyses of variance; **** P < 0.0001. Data are representative of three independent experiments ( d , middle and right, g ). Scale bars, 25 μm ( e ), 10 μm ( f ).
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a – d , HT - 29 cells with the indicated knockouts ( a ) or knockdowns ( b – d ) were treated with TSE (30 ng ml −1 hTNF, 0.2 µM SM-164, 5 µM emricasan) or TRAIL + SE (50 ng ml −1 , 0.2 µM SM-164, 5 µM emricasan) for 8 h, and cell death or cell viability was quantified using the indicated assays ( P = 1.39 × 10 −13 ( a ), P < 1 × 10 −15 ( b ), P < 1 × 10 −15 ( c ) and P = 6.82 × 10 −12 , P = 1.23 × 10 −11 ( d )). Cell lysates were analysed using reducing or non-reducing gel electrophoresis with the indicated antibodies. e , HT-29 cells with indicated stable short hairpin RNA (shRNA)-mediated knockdowns were treated with TSE for the indicated times, and cell death was assessed using CellToxGreen and time-lapse fluorescence confocal microscopy. f , As in e , except cell morphology was visualized by scanning electron microscopy. g , As in e , except the release of HMGB1 was analysed by immunoblotting. Data are plotted as the mean ± s.d., representative of n = 4 ( c ), n = 9 ( a , d ) or n = 12 ( b ); three independent experiments. Statistical analyses were performed using two-tailed t -tests and two-way analyses of variance; **** P < 0.0001. Data are representative of three independent experiments ( d , middle and right, g ). Scale bars, 25 μm ( e ), 10 μm ( f ).

Journal: Nature

Article Title: SIGLEC12 mediates plasma membrane rupture during necroptotic cell death

doi: 10.1038/s41586-025-09741-1

Figure Lengend Snippet: a – d , HT - 29 cells with the indicated knockouts ( a ) or knockdowns ( b – d ) were treated with TSE (30 ng ml −1 hTNF, 0.2 µM SM-164, 5 µM emricasan) or TRAIL + SE (50 ng ml −1 , 0.2 µM SM-164, 5 µM emricasan) for 8 h, and cell death or cell viability was quantified using the indicated assays ( P = 1.39 × 10 −13 ( a ), P < 1 × 10 −15 ( b ), P < 1 × 10 −15 ( c ) and P = 6.82 × 10 −12 , P = 1.23 × 10 −11 ( d )). Cell lysates were analysed using reducing or non-reducing gel electrophoresis with the indicated antibodies. e , HT-29 cells with indicated stable short hairpin RNA (shRNA)-mediated knockdowns were treated with TSE for the indicated times, and cell death was assessed using CellToxGreen and time-lapse fluorescence confocal microscopy. f , As in e , except cell morphology was visualized by scanning electron microscopy. g , As in e , except the release of HMGB1 was analysed by immunoblotting. Data are plotted as the mean ± s.d., representative of n = 4 ( c ), n = 9 ( a , d ) or n = 12 ( b ); three independent experiments. Statistical analyses were performed using two-tailed t -tests and two-way analyses of variance; **** P < 0.0001. Data are representative of three independent experiments ( d , middle and right, g ). Scale bars, 25 μm ( e ), 10 μm ( f ).

Article Snippet: The following reagents were used in this study: SM-164 (S7089), Nec-1s (S8641), GSK′872 (S8465), NSA (S8251), emricasan (S7775) and erastin (S7242) from SelleckChem; etoposide (E1383), α-ketoglutarate (349631), lipopolysaccharide (L4391), luminol (A8511), p -coumaric acid (C9008) and anti-FLAG M2 agarose beads (M8823) from Sigma-Aldrich; nigericin (11437) from the Cayman Chemical Company; Lipofectamine 3000 (L3000015), SuperSignal West Atto Ultimate Sensitivity Substrate (A38556) and Prolong Diamond Antifade Mountant with DAPI ( P36966 ) from Thermo Fisher Scientific; polyethylenimine (PEI; 24765-100) from Kyfora Bio; polybrene (TR-1003) from EMD Millipore; and 10X Tris/Glycine/SDS Electrophoresis Buffer (1610772), Tween 20 (1610781), Stacking Gel Buffer for PAGE (1610799), Resolving Gel Buffer for PAGE (1610798), Precision Plus Protein Dual Color Standards (1610394) and nitrocellulose membrane (1620115) from Bio-Rad.

Techniques: Nucleic Acid Electrophoresis, shRNA, Fluorescence, Confocal Microscopy, Electron Microscopy, Western Blot, Two Tailed Test