Anti his hrp

Purified collagen model peptides were coated onto a Nunc MaxiSorp (Thermo Fisher Scientific), 500 ng/well. Plates were washed after 1 h at RT with TBS-T 0.05% and free binding sites were blocked with 1% (w/v) BSA in TBS. The collagen type II library was first screened with single point measurements at a HSP47 concentration of 1 μM and positive hits further analyzed by titration experiments. In all titration experiments, the analyte exposure consisted of a 1:4 dilution series of HSP47, starting at 8 or 10 μM. HSP47 was detected by anti-His-HRP (1:10,000 in 1% BSA in TBS-T 0.05%, Miltenyi Biotec) and developed using freshly prepared TMB (3,3′,5,5′-Tetramethylbenzidin) solution. Assays were either stopped with 10% H₂SO₄ and absorption measured at 450 nm or continuously monitored at 370 nm every 20 min for 1 h. In latter cases, timepoints showing the highest signal-to-noise ratio were used for analysis. As a positive control collagen type I from rat tail collagen (Corning) was used. Results were analyzed using Gen5 (BioTek) and Origin 2018.

A Falcon 96-well clear flat bottom microplate was coated with PAM1.4 Fab-fragment, decorin (Sigma-Aldrich) or VAR2CSA full-length and individual domains in PBS (3μg/ml) overnight at 4°C. After incubating the plate was blocked for 1h at RT with PBS-T (PBS, 0.05% (v/v) tween-20, 1% BSA (w/v), pH. 7.4). Full-length VAR2CSA and shorter constructs (ID1-ID2a, DBL1-ID2a, DBL4 and DBL5) were added in a 2-fold dilution series in PBS from 50nM-1.56nM. Serum binding was assessed by adding diluted serum in PBS-T (1:200, 1:400, 1:800, and 1:1600) to plates coated with VAR2CSA. After incubation for 1h, plates were washed and added anti-HIS HRP (Miltenyi) diluted 1:3000 for VAR2CSA detection or anti-human Fc (1:5000) for IgG in serum detection. The plates were developed using TMB-plus2 (kementec) and the absorbance at 450nm was read.

Gel electrophoresis and western blots were done by standard procedures. The following antibodies were used: anti-c-myc-HRP (R951-25, Novex), anti-His-HRP (130-092-785, Miltenyi Biotec), mouse anti-α-tubulin (T9026, Sigma), goat anti-mouse-HRP (sc-2005, Santa Cruz, Dallas TX, USA), goat anti-rabbit HRP (sc-2004, Santa Cruz), rabbit anti-human AUF1 (07–260, Upstate/Merck Millipore, Billerica, MA), mouse anti-human HuR (sc-5261, Santa Cruz).