Hrp goat antirabbit

The western blot showed in Supplementary Fig. 2d was performed using the Bio-Rad Trans-Blot Turbo Transfer system (semidry blotting apparatus and ready-to-use membranes). Ten micrograms of protein lysates from all samples (HEK293T cells cultured in six-well plates and treated as described in Cell culture, transfections and cell line generation section, lysed with RIPA buffer) were ran for 1.5 h at 150 V on a 4–10% polyacrylamide gel in Tris-glycine buffer. The membrane was cut at roughly 70 kDa according to the ladder (Thermo Fisher, Page Ruler Prestained Plus, catalog no. 26619) and hybridized overnight at 4 °C with anti-GAPDH (Sigma, catalog no. G8795-200UL) and anti-HA (NEB, catalog no. 3724S), both diluted 1:1,000 in 5% milk TBS-T. Membranes were washed three times in TBS-T, hybridized for 1 h with horseradish peroxidase- (HRP-)conjugated secondary antibodies (HRP goat antirabbit, Dako, catalog no. P0448 and HRP goat antimouse, Invitrogen, catalog no. 31430, diluted 1:5,000), washed again and imaged using ECL detection (Thermo Fisher, catalog no. RPN2235) on a Vilber Fusion FX7 Edge imager with the Evolution-CaptEdge Fusion FX Edge (v.18.09) software.

Naïve and FVIII-exposed B cells as well as 413 cells were incubated with BDD FVIII (11.4 μg/ml), rFVIIIFc (14.7 μg/ml), goat anti-mouse IgG F(ab)₂ (αIgG F(ab)₂, 20 μg/ml, Southern Biotech) or whole goat anti-mouse IgG (αIgG, 20 μg/ml, Southern Biotech) for 30 min at 37°C. Cell lysates were then extracted and separated on an SDS PAGE gel, followed by transfer to nitrocellulose membrane (Bio Rad). Membranes were then blotted for phosphorylated SH2-containing inositol phosphatase (pSHIP, Cell Signaling Technology), SHIP (Santa Cruz Biotechnology), phosphorylated ERK (pERK, Cell Signaling Technology), ERK (Cell Signaling Technology) and actin (Abcam). Detection was carried out using horseradish peroxidase—conjugated (HRP) goat anti-rabbit (Dako) and goat anti-mouse (Southern Biotech) Ig followed by development with an enhanced chemiluminescence substrate (PerkinElmer). Densitometry analysis was performed using ImageJ (NIH) and ratios of phosphorylated to total protein were averaged for three different blots. No statistical analysis was carried out for these data due to the qualitative nature of the assay.