Hrp coupled secondary antibody

Analysis of changes in gene expression was performed by quantitative real time PCR (qRT‐PCR). RNA was isolated using the RNeasy plus kit (Qiagen). Five hundred nanograms of mRNA were reverse transcribed using qScript cDNA synthesis kit (Quanta Biosciences). The resulting cDNA was analyzed on a RotorGene Q cycler (Qiagen) using PerfeCTa SYBR Green FastMix (Quanta Biosciences). For primer sequences see Table 1. The cycling program was 30 s 95°C for denaturing, followed by 45 cycles of 5 s 95°C, 15 s 56°C, and 10 s 72°C. Data analysis was performed by normalizing values to ß‐actin as a house‐keeping gene and comparing expression to normoxia (21% O₂) exposure using the ΔΔCt method. Values generated by qPCR are given as “fold change compared to 21% O₂.”
Protein levels were analyzed by SDS‐PAGE and western blotting. Transferred proteins were detected by primary anti‐ACE/CD143 (R&D Systems cat.nr. AF1513), anti‐ACE2 (R&D Systems cat.nr.AF3437), anti‐eNOS (Cell Signaling Technology cat.nr. 9,586), anti‐ß‐Actin (Cell Signaling Technology cat.nr. 3,700) and according to species‐specific HRP‐coupled secondary antibodies (Rockland, USA). Bands were visualized by chemiluminescence (ECL Select Western blotting Detection Reagent; Amersham; GE Healthcare) using a Gel Documentation System (Vilber Lourmat).

HK-2 and CCD-1092Sk cells in monoculture were seeded in 24-well-plates or respectively in coculture as described in 2.2. The cells were incubated with media enriched with 50 mg/l ascorbic acid to support collagen synthesis and 50 mg/l ß-aminoproprionitrile to avoid collagen polymerization, which improves collagen determination. Collagen and fibronectin concentration was measured in the media according to and normalized to cellular protein content [25 (link)]. Antibodies against collagen III (1:1000) and fibronectin 1 (1:2000) were obtained from Biomol. HRP-coupled secondary antibodies (1:5000) were obtained from Cell Signaling (see Table 1).

Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, #10600003). Membranes were blocked in TBST + 5% BSA, incubated in primary antibody in TBST + 5% BSA o/n at 4 °C. After incubation with HRP-coupled secondary antibodies (Cell Signaling Technology, #7074, #7076 S), blots were subjected to ECL reaction. Images were acquired using ChemoStar (INTAS Science Imaging Instruments GmbH) and contrast was adjusted using ImageJ (version 1.52d). The band with the strongest signal was set to maximum (black). Quantification of Western blot signals was performed with ImageJ (version 1.52.d). Primary antibodies and dilutions were: C1qA (#11602-1-AP; 1:2000), C1qB (#16919-1-AP; 1:2000), C1qC (#66268-1-Ig; 1:4000), C1R (#17346-1-AP; 1:2000), C1S (#14554-1-AP; 1:4000), C3/C3b/C3 (#21337-1-AP; 1:1000), CFP (#17192-1-AP; 1:1000), CRP (#66250-1-Ig; 1:10000), F2 (#24295-1-AP; 1:5000), F7 (#23058-1-AP; 1:1000), FGG (#15841-1-AP; 1:2000), MBL2 (#24207-1-AP; 1:2000), SAP (#20773-1-AP; 1:2000) (all from Proteintech).
APOB (#sc-393636; 1:200) and MASP-1/3 (#sc-166815; 1:200) were from Santa Cruz Biotechnology.