Imagequant las 4000 luminescent image analyzer

The obtained protein fractions were analyzed using standard SDS-PAGE, followed by Coomassie Brilliant Blue R-250 (CBB, Sigma-Aldrich, St. Louis, MO, USA) or Western blot using polyclonal anti-mouse C-terminal AMCase^{33 (link)} or polyclonal pig anti-pepsin antibody (GeneTex, Irvine, CA, USA), followed by peroxidase-conjugated AffiniPure F (ab’)₂ Fragment Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or AffiniPure Donkey Anti-Goat IgG-HRP (Jackson ImmunoResearch laboratories). The immunoblots were analyzed and quantified by Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions.

Protein samples (20 μg) were separated by using 12% denaturing polyacrylamide gels, then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany) using a semi-dry transfer apparatus (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and immunoblotting was performed overnight at 4°C with the antibodies: p38 (Cell Signaling Technology, 1:1000), p-p38 (phospho T180 + Y182, Abcam, 1:1000), NF-κB (p65, Cell Signaling Technology, 1:1000), p-NF-κB p65 (phospho S536, Abcam, 1:1000), JNK (SAPK/JNK, Cell Signaling Technology, 1:1000), p-JNK (phospho T183 + Y185, Cell Signaling Technology, 1:1000), ERK1/2 (p44/42, Cell Signaling Technology, 1:1000), p-ERK (p-p44/42, phospho T202 + Y204, Cell Signaling Technology, 1:1000), and NLRP3 (Abcam, 1:2000), followed by incubation with secondary antibody conjugated to HRP (Abgent, San Diego, CA, USA, 1:10000 dilution). Meanwhile, β-actin (Cell Signaling Technology, 1:1000) was used to validate protein loading equivalence simultaneously. Signals were generated by enhanced chemiluminescence (Millipore, Germany) according to the manufacturer's protocol and detected by a Luminescent Image Analyzer (Image Quant LAS 4000, GE imagination at work, USA).

We analyzed the protein fractions using standard SDS-PAGE, followed by Coomassie Brilliant Blue R-250 (Sigma-Aldrich) or Western blot. Separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Merck Millipore, Tokyo, Japan), which was probed using polyclonal anti-pig pepsin antibody (GeneTex, Irvine, CA, USA) or polyclonal anti-pig Chia, followed by incubation with horseradish peroxidase conjugated secondary antibodies of AffiniPure Donkey Anti-Goat IgG-HRP (Jackson ImmunoResearch laboratories) or AffiniPure F (ab’)₂ Fragment Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). We analyzed and quantified the immunoblots using Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare, Piscataway, NJ, USA).

Primers for qPCR were designed based on Primer Express Software (Applied Biosystems) and were synthesized commercially (Sigma-Genosys, Sigma-Aldrich). The PCR reactions were performed in a final volume of 13 μl containing 2 x SYBR Green Master Mix (Brilliant II SYBR Green QPCR Master Mix, Agilent), 2.7 ng of mouse cDNA or appropriate dilutions of the external standards (see below), and 2.5 pmol of the primers for the three CLPs. The PCR reactions were performed using Mx3005P QPCR System (Agilent). The PCR program was as follows: 10 min of denaturation at 95°C, 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 1 min and polymerization at 72°C for 1 min. Melting curves were generated after amplification. The PCR products were electrophoresed on a 10% polyacrylamide gel and analyzed using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare). The nucleotide sequences of the primers that were used for the qPCR are shown in Supplementary Information (Additional file 1: Table S1). The Chit1, AMCase, pepsinogen C, GAPDH and β-actin primers have been previously reported [40 (link)].

The obtained protein fractions were subjected to standard SDS-PAGE, followed by SYPRO Ruby staining (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Healthcare) with All Blue (Bio-Rad Laboratories, Hercules, CA, USA) as the molecular weight marker.

About 3 μg C3b and 60 ng factor I (CompTech, USA) were incubated for 60 min at 37 °C with 0 to 160 ng HuCR1. After incubation, 2× prediluted NuPAGE LDS Sample Reducing Agent (Thermo Fisher Scientific, USA) was added at a 1/1 (v/v) ratio and heated at 60 °C for 15 min. Reduced samples were then run on 8% BisTris Plus SDS gels (Thermo Fisher Scientific, USA) to separate C3b α chain from its fragments. A Coomassie stain was then performed according to manufacturer's instructions, using the GelCode Blue Stain Reagent (Thermo Fisher Scientific, USA). For the quantification of the cofactor activity of the analyzed HuCR1 variants, stained gels were imaged using an ImageQuant LAS 4000 Luminescent Image Analyzer (GE Healthcare, USA), and the intact C3 α chain was quantified using GeneTools densitometry Software (SynGene, UK). For the calculation of uncleaved C3b α chain, a control sample (C3b incubated with factor I only) was run in parallel and set as 100% C3b α chain.