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sheep anti human tgn46  (Bio-Rad)
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Bio-Rad sheep anti human tgn46
Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
Sheep Anti Human Tgn46, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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clarity western ecl  (Bio-Rad)
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Bio-Rad clarity western ecl
Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
Clarity Western Ecl, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mini trans blot biorad apparatus  (Bio-Rad)
99
Bio-Rad mini trans blot biorad apparatus
Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
Mini Trans Blot Biorad Apparatus, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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trans blot turbotm mini nitrocellulose transfer packs  (Bio-Rad)
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Bio-Rad trans blot turbotm mini nitrocellulose transfer packs
Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
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silver stain plus kit  (Bio-Rad)
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Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
Silver Stain Plus Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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low fluorescence pvdf membrane  (Bio-Rad)
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Bio-Rad low fluorescence pvdf membrane
Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
Low Fluorescence Pvdf Membrane, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pvdf membrane  (Bio-Rad)
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Bio-Rad pvdf membrane
Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
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chemidoc imaging systems  (Bio-Rad)
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Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
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nitrocellulose membrane  (Bio-Rad)
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Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
Nitrocellulose Membrane, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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trans blot nitrocellulose membranes  (Bio-Rad)
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Bio-Rad trans blot nitrocellulose membranes
Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
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trans blot turbo transfer starter system  (Bio-Rad)
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Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
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polyvinylidene difluoride  (Bio-Rad)
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Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), <t>TGN46</t> (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.
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Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), TGN46 (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.

Journal: Cell death discovery

Article Title: Squaramides enhance NLRP3 inflammasome activation by lowering intracellular potassium.

doi: 10.1038/s41420-023-01756-9

Figure Lengend Snippet: Fig. 5 The effects of squaramides on other NLRP3 activation mechanisms. A BMDMs were pre-treated with 10 μM NVR compound, vehicle control (DMSO 0.5% v/v) or with the NFκB inhibitor Bay11-7082 (10 μM). After 15 minutes, Bay11-7082 was washed off and all cells were stimulated with 1 μg/mL LPS for 4 h. Cell lysates were probed for NLRP3 and pro-IL-1β induction. Western blots shown are representative of 3 biological repeats. B Cell treated as in (A). 4 hours post-LPS treatment cell culture supernatants were recovered and analysed for IL-6 content. Data correspond to 4 biological repeats. C COS7 cells were pre-treated with 10 μM NVR compound or vehicle control (DMSO 0.5% v/v) for 15 min and subsequently treated with 10 μM nigericin or vehicle control (EtOH 0.5%) for 90 min. Fixed cells were stained for Golgin97 (green), TGN46 (red) and nucleus (DAPI, blue). Images shown are representative of 4 biological repeats. Scale bar corresponds to 10 μm. D Pearson’s correlation coefficient (PCC) between Golgin97 and TGN46 for the cells treated in C. Data correspond to median +/− interquartile range from 4 biological repeats, where each datapoint is an individual field of view. Differences assessed by Kruskal-Wallis followed by Dunn’s post-test. ****p < 0.001. E LPS-primed BMDMs (1 μg/mL LPS; 4 h) were pre-treated with Cycloheximide (Chx, 10 μg/mL) or vehicle control (DMSO 0.5% v/v) for 30 min and subsequently stimulated with 10 μM nigericin in the presence of 10 μM NVR compounds or DMSO. After 90 min, culture supernatant was recovered and analysed for IL-1β content (left) or LDH release (right). Data correspond to mean +/−SD from 4 biological repeats. Differences assessed by two-way ANOVA followed by Dunnett’s post-test. ****p < 0.001. See also Fig. S4 for efficacy check of Chx.

Article Snippet: Coverslips were incubated with sheep anti-human TGN46 (Biorad, AHP500G) and rabbit anti-human Golgin97 (Abcam, ab84340) in PBS-Triton for 1 h at RT.

Techniques: Activation Assay, Control, Western Blot, Cell Culture, Staining