Azurespot analysis software

HaCaT cells (3 × 10⁵) were plated in 6-cm dishes and incubated with complete cell culture media which were pretreated with various conditions of He/Ar-CAPJ for 24 h. The treated cells were lysed with 100 μL RIPA reagent containing with protease and phosphatase inhibitors (Roch, Indianapolis, IN, United States). The cell lysates resolved by 10% SDS-PAGE and then performed the Western blot assay. The samples were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, United States) and immersed with 5% dehydrated skim milk to block non-specific protein binding. The membranes were incubated with primary antibodies as indicated in each experiment. The blots were then probed with the HRP-conjugated secondary antibody. The proteins of interest were detected using ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ, United States) and visualized by using Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA, United States). The signal intensity of each protein was quantified with UN-SCAN-IT software (Silk Scientific Corporation, Orem, UT, United States) as described previously (Lin et al., 2019 (link)). These data were expressed as the mean ± standard deviation determined from three independent experiments.

The spleen was collected 1 and 15 dpi, weighted and rapidly snap-frozen in liquid nitrogen, and stored at − 80 °C. Half-spleen was homogenized in ice-cold RIPA buffer with protease inhibitor cocktail (cOmplete; Roche, Switzerland). Samples were sonicated for 5 min, centrifuged, and supernatant collected for protein quantification. Bradford assay (BioRad, USA) was used for protein quantification. Samples were boiled at 100 °C for 5 min with Laemli buffer and β-mercaptoethanol. Twenty micrograms of total protein was loaded into a 15% SDS-Page gel and then transferred to a nitrocellulose membrane. After overnight incubation at 4 °C with primary antibodies: rabbit anti-TH (1:1000, Millipore, USA), mouse anti-βIII tubulin (1:1000, Promega, UK), mouse anti-βactin (1:1000, Abcam, UK), the secondary antibodies were incubated for 1 h at room temperature following dilutions: anti-rabbit-HRP (1:10.000, BioRad, USA) and anti-mouse-HRP (1:10.000, BioRad, USA). Antibody binding was assessed by chemiluminescence (ECL kit, BioRad, USA) in a Sapphire Biomolecular Imager (Azure Biosystems, USA). Band quantification was performed using the Azure Spot Analysis software (Azure Biosystems, USA) according to manufacturer’s instructions using β-actin as the loading control.

Each recombinant CdtA, CdtB, and CdtC was prepared and subjected to 12% SDS-PAGE, respectively. The gel was stained with Coomassie Brilliant Blue R-250 (Amresco, Solon, OH) for further analysis. AGS cells (5 × 10⁵) were exposed to CDT holotoxin with various concentrations for different time durations. The cell lysates were prepared to resolve by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were probed with primary antibodies: RAGE and HMGB1 (Abcam, Cambridge, UK), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Millipore, Temecula, CA). The proteins of interests were detected using the ECL Western Blotting Detection Reagent (GE Healthcare, Piscataway, NJ) and visualized by using Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, Dublin, CA).

Human plasma (collected
in tubes containing anticoagulant EDTA) was isolated from whole blood
as described elsewhere^{33 (link)} and incubated
with a fluorescently labeled probe at probe concentrations ranging
from 1 to 20 μM. Incubation was carried out in assay buffer
for 60 min at 37 °C. Plasma was incubated with the probe in a
total volume of 40 μL (20 μL of plasma and 20 μL
of probe) followed by reduction with 20 μL of 3 × SDS/DTT
for 5 min at 95 °C. The first well was loaded with 0.5 μL
of the protein marker PageRuler Prestained Protein Ladder (Thermo
Scientific), and then 5 μL of each sample was run on a 12% (w/v)
15-well gel. SDS–PAGE separation was performed at 200 V for
39 min followed by transfer to a nitrocellulose membrane (0.2 μm,
Bio-Rad) at 10 V for 60 min. The membrane was blocked with 2.5% BSA
in TBS-T for 60 min at room temperature. Next, the membrane was treated
with sheep antihuman polyclonal fXI antibody (Haematologic Technologies
Inc., PAHFXI-S, 1:1000) for 7 h followed by incubation with Alexa
Fluor 680 donkey antisheep secondary antibody (Life Technologies,
A21102, 2:10,000) for 1 h (both at room temperature). The membrane
was then scanned using an Azure Biosystems Sapphire Biomolecular Imager
and Azure Spot Analysis software for fXIa at 488 nm (for BODIPY detection)
and 658 nm (for antibody detection).