Nbt bcip substrate

Cell lysates were prepared in standard NP-40 lysis buffer (Abcam, USA) supplemented with proteinase and phosphatase inhibitors. Protein lysates were quantified using the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). Equal masses of total protein were separated on 4–12% SDS-polyacrylamide mini-gels, and blotted onto PVDF membranes (Millipore, USA). Membranes were subsequently blocked, incubated with primary antibodies, and incubated with secondary antibodies according to WesternBreeze Chromogenic Kit (Thermo Scientific, USA). Alkaline phosphatase was detected on the PVDF membranes using a ready-to-use BCIP/NBT substrate (Thermo Scientific, USA) for ready visualization of enzyme-linked antibodies. Rabbit anti-STAT1(149943), anti-phospho-STAT1(76493), anti-STAT3(91393), anti-phospho-STAT3 (9131), anti-phospho-ERK(19762), and anti-GAPDH (2118) antibodies were obtained from Cell Signaling (USA). Quantification of relative intensities was achieved by ImageJ analysis.

To qualitatively discern the reactivity of subjects’ sera with Sm-TSP-2, Western blot screening was performed with a selected set of blinded samples. Recombinant Sm-TSP-2 [13 (link),14 (link),18 (link)] was loaded (2 μg) under non-reducing and reducing (with addition of 2-mercaptoethanol, βME) conditions on a 4–20% SDS-PAGE gel (Invitrogen) at 135 V for 80 min. An irrelevant control recombinant protein (Na-GST-1, also expressed in a Pichia Pink system) was loaded as a negative control. Gels were then either stained with SimplyBlue™ (Thermo) or transferred on to 0.2 μm nitrocellulose membranes for 60 min at 30 V. Following transfer, the blots were stained with Ponceau S (Sigma) to confirm protein transfer and protein loads, followed by blocking with 2% milk diluted in 1X-PBST-20 (0.05%). Serum samples were then diluted at 1:100 in blocking buffer and incubated for 60 min at room temperature. Blots were washed 3 times with 1X-PBST-20 (0.05%) and then incubated with alkaline phosphatase secondary antibody, goat antihuman IgG (Southern Biotech) at a dilution of 1:1000 in blocking buffer. Following subsequent 3 washes with 1X-PBST-20 (0.05%), the blots were developed for 15 min with BCIP/NBT substrate (Invitrogen), then stopped with multiple washes with ultra-pure water. Blots were scanned together on a flatbed scanner (Epson) for visual documentation.

ELISpot for total IgG antibody secreting cells was performed as described previously (38 (link)). In brief, Milipore multiscreen plates (Milipore, MA) were coated with 2 μg/mL Goat Anti-mouse Ig (Southern Biotech). The following day, plates were washed and blocked with complete medium. CD19 magnetic bead (Miltenyi) enriched B cells (50,000 cells) were added to the plate and incubated for 18 hours at 37⁰C and 5% CO₂. Standard procedures using biotinylated anti-mouse IgG (Sigma) was followed with streptavidin-ALP (mABTech), and assay was developed using NBT/BCIP substrate (Thermo-Scientific), and analyzed using CTL Immunospot 5.0 software (Cellular Tech Ltd.). Data are represented as IgG antibody secreting cells (ASC) spots per 10⁶ cells (IgG ASC/10⁶ cells).