PDI inhibitors 16F16 (Sigma, St. Louis, MO, USA; SML0021), PACMA 31 (Sigma, St. Louis, MO, USA; SML0838), and di-tert-butyl nitroxide (DTBN, Sigma, St. Louis, MO, USA; 300721) were dissolved in DMSO and used at the indicated concentrations. Caco-2 cells grown in 96-well plates were washed once with MEM and then pretreated with the inhibitors, or DMSO as control, for 1 h at RT. After removal of the drugs, the cell monolayers were washed twice with MEM and then infected with the different serotypes of HAstV at an MOI of 0.025 for 1 h at 37 °C. After 16 hpi the cells were fixed and the virus infectivity was determined by a focus-forming unit (ffu) assay, as described above.
Pacma 31
Manufactured by Merck Group
Sourced in United States
PACMA 31 is a laboratory equipment product manufactured by Merck Group. It is designed for performing analytical procedures within a controlled environment. The core function of PACMA 31 is to provide a regulated and monitored space for conducting various laboratory analyses and experiments.
Automatically generated - may contain errors
5 protocols using pacma 31
1
Inhibition of HAstV Infection in Caco-2 Cells
2
Insulin Inhibition by PDI Modulators
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PDIs (400 nM) were solubilized in PBS pH 7.4 and then added to a solution containing 0.2 mM human insulin (Sigma–Aldrich), 2 mM EDTA, and 325 μM DTT. The reaction was monitored at 650 nm (turbidity due to precipitation of the product) for 1 h at 25 °C using a Spark multimode plate reader (Tecan). Increasing concentrations (0–100 μM) of the inhibitors 16F16 (Sigma–Aldrich) and PACMA-31 (Sigma–Aldrich) were incubated for 10 min with PDI before adding insulin. Data were analyzed using Origin 7.5 and graphed using Prism 9.0 (GraphPad Software).
3
Yeast Cell Binding Assay with Macrophages
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Yeast cells were washed once in PBS prior to introduction into wells with RAW 264.7 macrophages in DMEM (no FBS). Binding was allowed to proceed and was evaluated and quantified as described above for BAD-1-coated beads, except that prior to the last DMEM wash, yeast cells were briefly stained with Uvitex 3BSA (CIBA-Geigy, Basel, Switzerland) diluted 1:2,000 for contrast. The PDI inhibitors DTNB, Rutin, bisphenol, and PACMA31 were obtained from Sigma and dissolved at 100 mM in DMSO to make working stocks.
4
Müller Glia and R-Cognin Experiments
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For the Müller glia experiments, cells were incubated with 25 μM DAPT (Sigma) or the equivalent volume of DMSO starting from the equivalent time of 45-48 hpf, or 63 hpf.
For the R-Cognin experiments, cells were incubated with 5, 50, 100 or 200 μM PACMA31 (Sigma) or the highest equivalent volume of DMSO from the time of cell seeding onwards.
For the R-Cognin experiments, cells were incubated with 5, 50, 100 or 200 μM PACMA31 (Sigma) or the highest equivalent volume of DMSO from the time of cell seeding onwards.
5
Inhibition Assays for Cellular Processes
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PDI inhibitors (16F16, bacitracin, cystamine, cysteamine, hypotaurine, PACMA 31) and cycloheximide were purchased from Sigma-Aldrich (Saint-Louis, Missouri, USA), P1 was obtained from Tocris Bioscience and tunicamycin was purchased from Calbiochem (San Diego, CA, USA). These inhibitors were dissolved in DMSO (16F16/cystamine/cysteamine/PACMA 31/P1/tunicamycin/cycloheximide) or in cell culture medium (bacitracin/hypotaurine). Small RNA interference [siRNA] were obtained from Ambion (Austin, TX, USA) and plasmids from Origene (Rockville, MD, USA). All antibodies and siRNA used in this work have been described in tables included in the Supplementary data . These tables indicate providers and catalogue number of each antibody and also the sequences of all the siRNA (Table S5 ).
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