For immunoblotting analysis infected cell monolayers or media only control (mock infected) were washed in ice-cold PBS and lysed in RIPA Buffer (Teknova, Hollister, CA, USA) in the presence of a protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were resolved on NuPAGE 4–12% w/v Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes following manufacturer instructions. Immunodetection was performed using the following antibodies: Rabbit polyclonal Anti-PPP1CB using a dilution of 1:1000 (Cat# ab53315, Abcam, Cambridge, UK), mouse monoclonal antibody anti-α-Tubulin using a dilution of 1:3000 DM1A (Thermo Fisher Scientific, Waltham, MA, USA), and anti-CSFV E2 protein monoclonal antibody WH303 using a dilution of 1:1000 [32 (link)] and Pierce Goat Anti-Rabbit IgG peroxidase conjugated or Pierce Goat Anti-Mouse IgG peroxidase conjugated secondary antibody reagent (Thermo Fisher Scientific, Waltham, MA, USA). Western blots were imaged using an Azure C300 and analyzed with cSeries capture software (Azure Biosystems, Dublin, CA, USA).
Cseries capture software
Manufactured by Azure Biosystems
Sourced in United States
The CSeries capture software is a data acquisition and analysis tool designed to work with Azure Biosystems' laboratory equipment. It provides users with the ability to capture and record experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Thermo Fisher Scientific (1 mentions)
Azure c300, by Azure Biosystems (1 mentions)
Ripa buffer, by Teknova (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Iblot 2 gel transfer device, by Thermo Fisher Scientific (1 mentions)
Lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Azure c600 imaging system, by Azure Biosystems (1 mentions)
Bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Pierce ecl substrate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse igg, by GE Healthcare (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Azure c500, by Azure Biosystems (1 mentions)
Af1857, by R&D Systems (1 mentions)
Azure 600, by Azure Biosystems (1 mentions)
T6199, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
4 protocols using cseries capture software
1
Immunoblotting Analysis of CSFV Infection
2
Western Blot Analysis of FMDV Proteins
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The protein samples were mixed with 4× lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA, USA) and heated at 90 °C for 10 min. Samples were run on 4–12% gradient bis–tris gels (Invitrogen) and then transferred to a nitrocellulose membrane using the iBlot2 gel-transfer device (Invitrogen). The membranes were blocked in buffer [PBST; 10 mM sodium phosphate, 132 mM NaCl, 2.7 mM KCl, and 0.05% Tween-20, pH 7.4] for 1 h at 25 °C with shaking, washed three times with PBS-T for 10 min, and then incubated with a home-made primary antibody against FMDV VP1 (76.5E) and 3B (4G24) diluted 1/2000 in PBST at 4 °C overnight. The membranes were washed three times with PBS-T and incubated with HRP-conjugated goat anti-mouse secondary antibody (Invitrogen) diluted 1/4000 in PBST for 1 h at RT. Proteins were detected with Pierce ECL Substrate (Invitrogen) using an Azure C600 imaging system and cSeries Capture Software (Azure Biosystems, Dublin, CA, USA).
3
Comprehensive Multiomics Data Analysis Pipeline
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Data were collected using Windows 10, Microsoft Excel for Office 365, StepOne Software v2.3, Zeiss Zen software 2.6, Epson scan 3.9.4.7US, and Azure biosystems cSeries capture software 1.9.7.0802. The data were analyzed by Windows 10, Microsoft Excel for Office 365, Microsoft Word for Office 365, GraphPad Prism 8.4.2, StepOne Software v2.3, NIH ImageJ 1.52a, EgdeR package 3.24.1, Enrichr (https://amp.pharm.mssm.edu/Enrichr/ ) version January 7th, 2020, GeneXplain version 4.8, ingenuity pathway analysis (Build version: 430520 M Content version: 31813283), Overlap stats (nemates.org/MA/progs/overlap_stats.html) version 2019, Sequest (SRF v.5), and HTSeq 0.9.1.
4
Western Blot Analysis of LCN2/NGAL in RPE/Choroid
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Protein was extracted from the RPE/choroid, according to a previously described methodology [28 (link)] and protein concentrations were measured by Pierce™ BCA protein assay kit (Thermo Fisher Scientific). Subsequently, 10 μg of protein was loaded into each well, separated on a 4–12% Bis-Tris SDS-PAGE gel (NuPAGE, Invitrogen), and transferred to a polyvinylidene fluoride membrane (Bio-Rad). After blocking with 5% bovine serum albumin (BSA, Sigma) in Tris-buffered saline (TBS, Bio-Rad), membranes were incubated overnight with primary antibodies: anti-mouse LCN2/NGAL (1:1000; catalog# AF1857, R&D Systems), or anti-α-Tubulin (1:1000; catalog# T6199, Millipore Sigma), used as a loading control. After three washes with 0.1% Tween-20 in TBS, incubated for 1 h with appropriate secondary antibody, either horseradish peroxidase (HRP)-conjugated anti-goat IgG (1:7500; Jackson ImmunoResearch), or HRP-conjugated anti-mouse IgG (1:8000; GE healthcare Lifesciences). Membranes were developed using SuperSignal™ West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific). Images were acquired using an Azure c500 or Azure 600 gel imager with cSeries Capture Software (Azure Biosystems).
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