Cnbr activated sepharose

Fragments of the ZC3H11 open reading frame (first 104a.a., 119a.a., 136a.a.) were cloned into pQEA38vector after His₁₀-tag, between KpnI and HindIII sites. Bacteria (E.coli strain Rosetta pLysS, Novagen) were grown at room temperature to an OD600 of 0.6, induced with 0.25mM isopropyl β-D-1-thiogalactopyranoside and incubated at the same temperature for five hours before harvesting. Recombinant proteins were purified with Ni-NTA Agarose (QIAGEN) following the manufacturers’ instructions [20 (link)]. Buffer in protein samples was exchanged to PBS. Rabbits were immunized with His₁₀-ZC3H11 (119a.a.) according to standard procedures (Charles River Laboratories, Kisslegg, Germany). Polyclonal antibodies were affinity-purified from crude anti-serum using His₁₀-ZC3H11 (119a.a.) fragment coupled to CNBR-activated Sepharose (GE Healthcare).

Peptides were purified as previously described (23 (link), 24 (link)). About 1 × 10⁹ cells were lysed in tris buffer (TB: 50 mM Tris/HCl (pH 7.6), 150 mM NaCl) with 1% Igepal-CA630 supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail Tablet, Hoffman-La Roche, Basel, Switzerland) at 4°C for 2 h. Lysates were centrifuged for 10 min at 1,300 × g, and supernatants were ultra-centrifuged for 1 h at 100,000 × g. The soluble fraction was precleared using a Tris-blocked sepharose column (CNBr-activated sepharose, GE Healthcare Bio-Sciences AB, Sweden). The flow-through was incubated overnight at 4°C with B8.11.2-sepharose and washed as follows: first, with 50 ml of TB, 0.5% Igepal-CA630; then, with 200 ml of TB; and, finally, with 500 ml of Tris–HCl pH 7.6, 5 mM NaCl. HLA-DR–peptide complexes were eluted in 0.1% trifluoroacetic acid. Protein-containing fractions were determined by the Bradford method, pooled and concentrated in a SpeedVac (Thermo Fisher scientific, Waltham, MA, USA). Peptides were purified by ultra-filtration in a Centricon-10 device (Millipore, Ireland, Ltd.). Retained material, containing HLA-DRα and β chains are shown in Figure S1 in Supplementary Material.

C1q was purified from freshly thawed plasma, as published earlier (26 (link)). Briefly, plasma was made 5mM EDTA, pH 7.5, and centrifuged at 12,000 × g to remove aggregated lipids. The plasma was then incubated with non-immune IgG coupled to CNBr-activated Sepharose (GE Healthcare, UK) for 1 h at 4°C. The plasma was filtered through a sintered glass funnel, and C1q bound to IgG–Sepharose was then washed extensively with 10 mM HEPES, 140 mM NaCl, 0.5 mM EDTA, and pH 7.0. C1q was eluted with N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer (100 mM CAPS, 1 M NaCl, 0.5 mM EDTA, pH 11). The eluted C1q was then passed through a HiTrap Protein G column (GE Healthcare) to remove IgG contaminants and dialyzed against the washing buffer.

Polyclonal antibodies against ForA and Rac1A were raised by immunizing a female New Zealand white rabbit with either recombinant GST-tagged ForA-FH3L or GST-tagged Rac1A following standard protocols. Subsequently, the polyclonal antibodies were affinity purified after coupling of the same antigens to CNBr-activated Sepharose (GE Healthcare). Immunoblotting was performed according to standard protocols using undiluted hybridoma supernatants of IQGAP1-specific mAb 216-394-1 (ref. 30 (link), GFP-specific mAb 264-449-2 (ref. 30 (link)), myosin-II-specific mAb 56-396-5 (ref. 64 (link)), Porin-specific mAb 70-100-1 (ref. 65 (link)) or polyclonal anti-Rac1A and anti-ForA antibodies (1:250 dilution). Alexa488-conjugated nanobodies were from Chromotek (1:200 dilution; #gba488) and polyclonal anti-myosin IIA antibodies were from Thermo Scientific (1:100 dilution; #PA5-17025). Primary antibodies in immunoblots were visualized with phosphatase-coupled anti-mouse (1:1,000, #115-055-62; Dianova) or anti-rabbit IgG (1:1,000; #111-055-046; Dianova). Uncropped scans of the most important immunoblots are shown in Supplementary Fig. 6. For immunohistochemistry, secondary Alexa-555-conjugated goat-anti-rabbit polyclonal antibodies (1:1,000 dilution; #A21429; Invitrogen) were used.