Cells were incubated in complete or starvation media in the absence and presence of 100 nM bafilomycin A1 (BafA1) (ThermoFisher scientific, J61835.MCR) for 3 h and subjected to immunoblotting using the mCherry antibody (Abcam, ab125096) with a ratio of 1:1000, the β-actin antibody (Sigma, A5441-100UL) with a ratio of 1:10,000, and the LC3 antibody (Novus Biologicals, NB100-2220) with a ratio of 1:5000. To quantify the LC3-II expression level, western blot experiments were performed 4 times. The signal intensity of LC3-II was measured using Image Studio version 5 software (LI-COR Biotechnology), then normalized to the intensity of β-actin of the same sample. To calculate the relative LC3-II level, the LC3-II value of Atg3−/− MEFs expressing mCherry-hAtg3 in starvation medium with BafA1 was set to 1. The autophagic flux was calculated by subtracting the LC3-II value in the absence of BafA1 from that in the presence of BafA1 in starvation medium, and the values were then normalized with the value of Atg3−/− MEFs expressing mCherry-hAtg3 being set as 1. The data were analyzed by one-way ANOVA test followed by Turkey’s multiple comparisons test using GraphPad Prism7.0.
A5441 100ul
Manufactured by Merck Group
Sourced in United States
A5441-100UL is a laboratory product offered by Merck Group. It is a solution used for various research and analytical applications. The product specifications and details are not available for an unbiased and factual description without interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Nb100 2220, by Novus Biologicals (2 mentions)
Image studio version 5, by LI COR (2 mentions)
Prism 7, by GraphPad (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Multi gauge software program, by Fujifilm (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Active caspase 3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Rabbit anti human insulin, by Cell Signaling Technology (1 mentions)
A9044 2ml, by Merck Group (1 mentions)
Ab 110305, by Abcam (1 mentions)
Ab118470, by Abcam (1 mentions)
Pa1 16893, by Thermo Fisher Scientific (1 mentions)
5 protocols using a5441 100ul
1
Measuring Autophagic Flux in Cells
2
Quantifying Autophagy Flux via LC3-II
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Cells were incubated in complete media or starvation media (amino-acid and serum free) in the presence or absence of 100 nM bafilomycin A1 (BafA1) for 3 h and subjected to immunoblotting using the mCherry antibody (Abcam, ab125096) with a ratio of 1:1000, the β-actin antibody (Sigma, A5441-100UL) with a ratio of 1:10,000, and the LC3 antibody (Novus Biologicals, NB100-2220) with a ratio of 1:5,000. To quantify the LC3-II level, the experiments were repeated six times. The signal intensity of LC3-II was measured using Image Studio version 5 software (LI-COR Biotechnology), then normalized to the intensity of β-actin of the same sample. To calculate the relative LC3-II level, the LC3-II value of Atg3−/− MEFs expressing mCherry-hAtg3 in starvation medium with BafA1 was set to be 1. The autophagic flux was calculated by subtracting the LC3-II value in the absence of BafA1 from that in the presence of BafA1 in complete (basal) or starvation (induced) medium. All the values were then normalized with the value of Atg3−/− MEFs expressing mCherry-hAtg3 being set as 1. The data were analyzed by one-way ANOVA test followed by Dunnett’s multiple comparisons test using GraphPad Prism7.0.
3
Western Blotting Analysis of Fibroblast Markers
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Western blotting was performed as previously described.14 (link) Briefly, the samples were separated by electrophoresis and the membranes were incubated at 25°C for 1 h with primary antibodies against S100A4, a fibroblast-specific marker (1:1000; RB-1804-A0, Thermo Fisher Scientific, Waltham, MA, USA), collagen type I (1:1000; 234,167, Millipore, Billerica, MA, USA), cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-9 (1:1000; Cell Signaling Technology), or β-actin as the loading control (1:5000; A5441-100UL, Sigma-Aldrich Corp., St. Louis, MO, USA). The membranes were then treated with a horseradish peroxidase-conjugated secondary antibody (1:1000; Novex, Frederick, MD, USA). Images of the membranes were acquired with the Multi-Gauge Software Program (Fujifilm, Greenwood, SC, USA).
4
Betatrophin and Insulin Protein Analysis
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Cell lysates (50 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences, Uppsala, Sweden). After blocking with 5% skim milk, the membranes were then blotted with the indicated primary antibodies: mouse anti-human betatrophin antibody (1:200; AG-25B-0033-C100; Adipogen, San Diego, CA, USA), rabbit anti-human insulin (1:200; Cat. no. 3014; Cell Signaling Technology, Danvers, MA, USA), active caspase-3 (1:200; Cat. no. 9661S; Cell Signaling Technology), mouse anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5,000; ab110305; Abcam, Cambridge, UK), or mouse anti-human β-actin (1:10,000; A5441-100UL; Sigma-Aldrich, St. Louis, MO, USA). After washing, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h: either goat anti-rabbit IgG (1:8,000; AS10 668; Epitomics Inc., Burlingame, CA, USA) or rabbit anti-mouse IgG (1:10,000; A9044-2ML; Sigma-Aldrich). The protein intensity was determined by ECL chemiluminescence reagent (PerkinElmer Life Sciences, Inc., Waltham, MA, USA), and their intensities were quantitatively measured by densitom-etry (LabWorks, UVP Inc., Upland, CA, USA).
5
Protein Level Detection of Ferroptosis Markers
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To detect the protein level alteration of ferrotosis-related molecules, western blotting was performed. The cell samples were lysed by RIPA buffer containing 0.1% SDS. After running the SDS–PAGE, the protein samples were transferred onto the PVDF membrane, and followed by antibody incubation. The antibodies were listed as follows: rabbit anti-SLC7A11 (1:1000, PA1-16893, Invitrogen, USA), rabbit anti-ALXOE3 (1:800, ab118470, Abcam, USA), and mouse anti-β-actin (1:5000, A5441-100UL, Sigma, USA). The blots were explored with ECL, and the immunoreactive signals were generated in a luminescence detection system using horseradish peroxidase-labeled secondary antibody.
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