Uvp biodoc it system

Western blotting was performed using standard methods. Cells were washed with PBS and then lysed in radioimmunoprecipitation assay lysis buffer; cellular protein lysates were isolated using a protein extraction kit (Qiagen, Carlsbad, CA, USA) and quantified using the Bradford protein assay kit (Carlsbad, CA, USA). After preparing the whole cell lysate, the proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with Tris-buffered saline and skimmed milk. These PVDF membranes were then probed with respective primary antibodies, followed by the secondary antibody. The commercial antibodies are described in Supplementary Table S1. Images were captured and analyzed using the UVP BioDoc-It system (Upland, CA, USA).

In order to compare protein profiles of deiminated proteins between E. coli and E. coli OMVs, total protein lysates and IP protein eluates were subjected to Western blot analysis. Briefly, samples were boiled for 5 min at 100°C in 2x Laemmli sample buffer (BioRad, U.K.). Protein (20 μg per sample) was separated by SDS-PAGE using 4–20% Mini-Protean TGX protein gels (BioRad, U.K) and transferred to nitrocellulose membranes. Even loading was assessed using Ponceau S staining (Sigma, U.K.) and membranes were then blocked in 5% bovine serum albumin (BSA) in Tris buffered saline with 0.1% Tween20 (TBS-T) for 1 h, followed by incubation at 4 °C overnight with the primary antibodies: pan-deimination antibody F95 (1:2,000; MABN328), PAD2 (1:1,000; ab50257), PAD3 (1:1,000; ab169479) and PAD4 (1:1,000; ab96758), respectively. Membranes were washed three times in TBS-T, incubated at room temperature for 1 h with HRP-conjugated secondary antibodies: anti-mouse IgM and anti-rabbit IgG, respectively (both 1:4,000; BioRad, U.K.), followed by six 10 min washes in TBS-T, before visualization using ECL (Amersham, U.K.) and the UVP BioDoc-IT™ System (U.K.).

PCR amplifications were initially performed on bulked DNA of the same 48 samples from each population (ATF0 and ATF5) to verify the specificity of the 11 amplicons. PCR amplifications were conducted in a total volume of 25 μL in 0.2 mL PCR strips containing 2.5 μL of 10X PCR buffer, 1 μL each of 5 μM primers, 0.5 μL of 10 mM dNTP (Roche Diagnostics, Laval, QC, Canada), 0.5 μL of 5 PRIME GmbH Taq Polymerase 5 U/μL (Inter Medico, Markham, ON, Canada) and 5 μL of 10 ng/μL DNA (50 ng). PCR amplifications were performed on an Eppendorf Mastercycler ep System (Eppendorf Canada, Mississauga, ON, Canada) as follows: 3 min of initial denaturation at 94°C; followed by 40 cycles at 94°C for 1 min, annealing at Tm for 1 min, extension at 72°C for 1 min; and final extension at 72°C for 7 min. The specificity of amplification was confirmed by running 20 μL of each reaction for 2–3 h at 70 V on a 2% agarose gel stained with ethidium bromide and products were visualized using a UVP BioDoc-It system (UVP, Upland, CA, USA). DNA fragments were recovered from agarose gels using the QIAquick gel extraction kit (QIAGEN Inc., Mississauga, ON, Canada). A consensus sequence of each amplicon was obtained by bidirectional Sanger sequencing on an ABI 3130xl DNA Sequencer (Plateforme d’analyses génomiques, IBIS, Laval University, Quebec City, QC, Canada).