Anti erm

10⁶ wt and hem1^−/− DCs were spun down and lysed in 50 µl 1× lysis buffer (1 ml 10× radioimmunoprecipitation assay (RIPA) buffer, New England Biolabs; 9 ml H₂O, 1 tablet PhosStop, and 1 tablet protease inhibitor mini, both Merck). Lysates were spun at full speed for 10 min at 4°C and supernatants were transferred to new tubes. Equal volumes of 2× loading buffer (4× lithium dodecyl sulfate (LDS) buffer, 10× sample-reducing agent) were added. Two lanes were run for each WT or hem1^−/− sample. Per lane, 15 µl were denatured at 99°C for 5 min. Samples were loaded on gels (NuPage 4–12% Bis-Tris Protein gels, Thermo Fisher Scientific) and run with 180 V. After protein transfer, membranes were cut in a way to allow for separate incubation with anti-ERM (1:1,000; Cell Signaling Technology, #3142), anti-pERM (1:1,000; Cell Signaling Technology, #3141), and anti-GAPDH (1:10,000; Abcam, ab125247) antibodies. Membranes were blocked with 5% BSA in 1× TBS with Tween 20 (TBS-T) for 1 h at RT, followed by incubation with the respective antibodies overnight at 4°C. Membranes were washed three times for 10 min with 1× TBS-T and then incubated with HRP-coupled secondary antibodies for 1 h at RT. Membranes were washed again three times with 1× TBS-T, incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and imaged on an Amersham imager 600 (GE Healthcare).