Assays

Cells were collected and lysed in urea buffer (9 M Urea, 50 mM Tris pH 8, 150 mM NaCl, 1x Roche protease inhibitor cocktail) followed by short sonification. Samples were cleared by centrifugation and protein amounts were adapted. Protein reduction was performed with dithiothreitol (DTT; 5 mM final) for 25 min at 56°C and protein alkylation by the addition of iodoacetamide (14 mM final) for 30 min at room temperature. Protein mixtures were quenched with DTT and diluted 1:5 with 1 M Tris-Hcl, pH ...

Samples were processed and analyszed as described in Behrends et al., Nature 2010.

Anti-HA-immunoprecipitation was performed as previously described (Behrends et al, 2010; Jung et al, 2015; Jung et al, 2017; Sowa et al, 2009). Summarily, expression of TAPL-HA and coreTAPL-HA was induced by addition of 4 µg/ml doxycycline for 24 h in HeLa Flp-In T-REx cells. Parental non-transfected HeLa Flp-In T-REx cells were used as negative control. For each sample, 6.4 x 107 cells were harvested, frozen in liquid nitrogen and stored at -80 °C. Cells were lysed in 3 ml MCLB buffer (50 mM ...

Cells (4x 15 cm dishes) were harvested and lysed with 3 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40 (NP40) and EDTA-free protease inhibitor cocktail tablets). Centrifugation-cleared lysates (13,000 rpm) were filtered through 0.45 µm spin filters (Millipore Ultrafree-CL) and immunoprecipitated with 60 µl anti-HA resin. Resin containing immune complexes were washed five times with lysis buffer followed by five PBS washes, and elution with 150 µl of 250 mg/ml HA peptide in ...