Roti block reagent

Peptides were dotted in a decreasing concentration pattern (0.33, 0.165, 0.083, 0.041, 0.021 mg/ml) on a nitrocellulose membrane (0.45 μm, Merck KGaA, Darmstadt, Germany), blocked with Roti-Block reagent (Roth, Karlsruhe, Germany) for 1 h at RT, followed by an overnight incubation with 1 μg/ml nAbs/IVIg solution in Roti at 4 °C. The membrane was washed in TBST (Tris-buffered saline with 0.05% Tween 20) and incubated with 1:500,000 HRP-conjugated anti-human detection antibody (Thermo Fisher Scientific Inc., Rockford, USA). Control stainings were performed by using the secondary antibody alone. Visualization was performed by using a HRP visualization kit (Super Signal West Dura Extended Duration Substrate, Thermo Fisher Scientific Inc., Rockford, USA) followed by exposure to X-ray film.

CLIP2 knockdown on protein level was verified using Western Blot analysis. Cells were washed once with cold 1xPBS and lysed using RIPA lysis buffer containing protease (Complete; Roche, Mannheim, Germany) and phosphatase inhibitor cocktails (PhosStop; Roche, Mannheim, Germany). 20 μg (knockdown) or 10 μg (overexpression) of the whole cell lysate was subjected to 10% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P; Millipore, Billerica, MA, USA). Roti-Block reagent (Carl Roth, Karlsruhe, Germany) was used for blocking. The membranes were incubated with anti-CLIP2 polyclonal rabbit antibody (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with the corresponding secondary antibody signals were visualized with the enhanced chemiluminescence system (Amersham ECL Plus Western blotting detection system; GE Healthcare, Chalfont St. Giles, UK).