Whole cell lysates from HEK 293T, HeLa and MDCK cells stably expressing Panx2-HA, Panx2-WT, or Panx1-WT proteins were incubated with 10 units of calf intestinal alkaline phosphatase (CIAP) for 5 h at 37°C; or with 1500 units of N-glycosidase F (PNGase F) (New England Biolabs, Beverly, MA) for 5 h at 37°C at pH 7.5 following procedures we used in Boassa et al. (2007 (link)) or 50 μM pan-caspase inhibitor Z-VAD-OMe-FMK for 5 h at 37°C (Millipore, Billerica, MA) following a procedures described in Penuela et al. (2014 (link)). Samples were boiled for 5 min, and western blotted as described above.
Z vad ome fmk
Manufactured by Merck Group
Sourced in Morocco
Z-VAD-OMe-FMK is a broad-spectrum caspase inhibitor that can be used in cell culture experiments to study the role of apoptosis in various biological processes. It functions by irreversibly binding to and inhibiting the activity of multiple caspase enzymes.
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Lab products found in correlation
Ciliobrevin d, by Merck Group (1 mentions)
Thymidine, by Merck Group (1 mentions)
Fas ligand, by R&D Systems (1 mentions)
Thioglycolate, by Merck Group (1 mentions)
Isotype matched control antibody, by BioLegend (1 mentions)
Il 1r, by BioLegend (1 mentions)
Il 1α, by R&D Systems (1 mentions)
1 hydroxypyridine 2 thione zinc salt, by Merck Group (1 mentions)
Pyrithione 2 mercaptopyridine n oxide sodium salt, by Merck Group (1 mentions)
4 protocols using z vad ome fmk
1
Post-translational Modifications of Panx Proteins
2
Live Cell Imaging: Cell Cycle Regulation
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For live cell imaging, cell cycle progression was blocked with 2.5 mM thymidine (Sigma, #T1895) for 24 h. After three washes with PBS, cells were released into fresh media for 10 h. For inhibitor experiments, cells were arrested in prometaphase with 5 µM S-trityl-L-cysteine (STLC, Sigma #164739) for 15 h, and Plk1 inhibitor BI2536 or 50 µM dynein inhibitor Ciliobrevin D (#250401, Millipore) was added for 1 h more. 5 µM of Cdk1 inhibitor RO-3336 (Millipore, #217699) was used to trigger mitotic exit. Z-VAD (OMe)-FMK was used for apoptosis inhibition (Millipore, #627610).
3
Humoral Factors Modulate Bone Marrow Dynamics
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To assess the contributions made by humoral factors, 5-wk-old CAG-eGFP mice were treated with TPO (Sigma-Aldrich) or IL-1α (R&D Systems or BioLegend) at dosages of 10 µg/mouse subcutaneously (SC) daily for 5 d or 70 µg/mouse SC daily for 3 d. The mice were visualized and examined at 6-wk-old, 7 d after the first treatment. Some mice were also treated with neutralizing antibodies against IL-1α (R&D Systems or BioLegend), IL-1R (BioLegend), isotype-matched control antibody (BioLegend), or Fas Ligand (5 µg/mouse i.v.; R&D Systems). All antibodies were administered at 100 µg/mouse i.p. daily for 3 d.
To assess the effect of acute inflammation, thioglycolate (Sigma-Aldrich) was administered i.p. (3 ml of 3% solution/mouse once). To examine the effect of caspase inhibition, mice were treated with the pan-caspase inhibitor Z-VAD (OMe)-FMK (3 mg/kg IP daily for 5 d; Merck Millipore). Clophosome (200 µl/mouse; FormuMax Scientific Inc.) was administered i.p. to deplete macrophages.
To obtain chimeric mice, 6-wk-old WT mice were lethally irradiated at a dose of 9.5 Gy, after which they were transplanted with BM total cells from age-matched IL-1R−/−/CAG-eGFP, IL-1α−/−/CAG-eGFP, or control (IL-1R+/+/CAG-eGFP or IL-1α+/+/CAG-eGFP) mice. 6 wk after transplantation, BM dynamics were scored.
To assess the effect of acute inflammation, thioglycolate (Sigma-Aldrich) was administered i.p. (3 ml of 3% solution/mouse once). To examine the effect of caspase inhibition, mice were treated with the pan-caspase inhibitor Z-VAD (OMe)-FMK (3 mg/kg IP daily for 5 d; Merck Millipore). Clophosome (200 µl/mouse; FormuMax Scientific Inc.) was administered i.p. to deplete macrophages.
To obtain chimeric mice, 6-wk-old WT mice were lethally irradiated at a dose of 9.5 Gy, after which they were transplanted with BM total cells from age-matched IL-1R−/−/CAG-eGFP, IL-1α−/−/CAG-eGFP, or control (IL-1R+/+/CAG-eGFP or IL-1α+/+/CAG-eGFP) mice. 6 wk after transplantation, BM dynamics were scored.
4
Utilizing Zinc-Pyridine Complexes and Caspase Inhibitor
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