Af902

Antibodies against complement components C1s (Sheep Anti‐human C1s, AF2060, and mouse anti‐human C1s MAB2060) and C1r antibodies (AF1807‐SP) and antibodies recognizing human MMP‐9 (AF911) and human MMP‐2 (AF902) were purchased from R&D Systems (Minneapolis, MN, USA). HRP‐conjugated antibodies were purchased from Jackson Human Research (Cambridgeshire, UK). Recombinant C1s (H00000716‐P01) was from Abnova (Taipei City, Taiwan). Gelatin from bovine skin, type B (G9391) for zymography analysis was from Sigma (Sigma‐Aldrich, St Louis, MO, USA). EDTA, protein G‐Sepharose 4 Fast Flow (71‐7083‐00) and gelatin‐Sepharose 4B (17‐0956‐01) were purchased from GE Healthcare (Chicago, IL, USA), Coomassie brilliant blue (B0149) was from Sigma. EDTA (VWR‐ 20302.260) was from BDH‐chemicals (Brooklyn, NY, USA).

Total protein was harvested using an assay kit (Beyotime Biotechnology, Shanghai, China). Bio-Rad DC Protein Assay Kit (Guangzhou Ewell Bio-technology Co., Ltd., Guangdong, China) was used for protein quantification. Each sample was added with sodium dodecyl sulfate (SDS) loading buffer, boiled for 10 min in boiling water, and 20 μg protein sample was applied to a 10% SDS-polyacrylamide gel. Then the protein was transferred onto polyvinylidene fluoride membrane and immersed in 1 × Tris-Buffered Saline Tween-20 (TBST) containing 5% skimmed milk powder to block non-specific binding sites. The membrane was then incubated overnight at 4 °C with diluted primary antibody, i.e. one of the rabbit antibodies LPA1 (R&D system, Minneapolis, MN, USA, AF9963, 1:2000), MCM2 (R&D system, AF5778, 1:2000), MMP2 (R&D system, AF902, 1:2000), MMP9 (R&D system, AF909, 1:2000), VEGF (R&D system, AF-493-NA, 1:2000), and GAPDH (R&D system, AF5718, 1:2000). Then the membrane was incubated with secondary goat anti-rabbit anti-immunoglobulin G (IgG) (R&D system, AB-105-C, 1:20,000). Exposure was carried out with an enhanced chemiluminescence. Gray value of each protein was determined by Image J software (NIH free software, Bethesda, MD, USA). The original immunoblotting bands are shown in Additional file 1.

MMP expression was analyzed by immunoblot in conditioned medium of LX‐2 cells, generated by cultivating the cells in serum‐deprived medium for 48 h, followed by protein concentration using vivaspin centrifugal concentrator columns (MWCO 3,0 kD, Sartorius GmbH). Protein contents were analyzed according to Bradford using Rotiquant (Roth, Karlsruhe, Germany). Either 20 or 40 μg of protein was loaded on 10% polyacrylamide gels and subsequently blotted on PVDF membranes. For the detection of specific proteins, the following antibodies were used: anti‐MMP‐2 (AF902, 2 μg·mL⁻¹; R&D Systems); anti‐MMP‐9 (MAB‐911, 4 μg·mL⁻¹, R&D); MMP‐7 (IMG‐3873; 0.5 μg·mL⁻¹, IMGENEX); MT1‐MMP/MMP‐14 (D1E4, 1 : 1000, Epitomics); TIMP‐2 (MAB971, 1 : 500, R&D). Protein contents were visualized on a ChemiDoc MP system using the imagelab software for quantification (both from Bio‐Rad Laboratories GmbH, Munich, Germany). For the demonstration of equal loading, the membranes were subsequently stained with Ponceau S (Sigma‐Aldrich). MMP‐2 and ‐9 activity was determined using gelatine containing zymography gels (Thermo Fisher Scientific, Karlsbad, CA, USA) as previously described [41 (link)].