Las 4000 mini bioimager

Samples were homogenized under liquid nitrogen to compare YFP-tagged TTG1 in the overexpression lines OE01-03 and OE19-21. 150 µl of lysis buffer (Kirik et al., 2007 (link)) were added to the powder and incubated for 30 min at 4 °C (rotating). 100 µl of the supernatant following centrifugation were mixed with 100 µl 2x Laemmli, boiled for 10 min at 95 °C and centrifuged for 1 min at 10 600 g. Samples were separated on SDS-PAGE gels, subsequently blotted and immunodetected (α-GFP (IgG1K, Roche), α-mouse (Jackson ImmunoResearch, http://www.jacksonimmuno.com)). After GFP detection using the SuperSignal® West Femto Maximum Sensitivity Substrate (ThermoFisher) and a LAS-4000 Mini bioimager (GE Healthcare Life Sciences (formerly Fuji), http://www.gelifesciences.com), blots were stripped as suggested by Abcam (http://www.abcam.com/ps/pdf/protocols/stripping%20for%20reprobing.pdf) using mild stripping buffer (1L: 15 g glycine, 1 g SDS, 10 ml Tween20, pH to 2.2) and re-probed with α-histone H3 (ab1791, Abcam, http://www.abcam.com) and α-rabbit (A6154, Sigma-Aldrich, http://www.sigmaaldrich.com).

For separating nuclear-enriched protein extracts by SDS-PAGE, equal volumes of nuclear-enriched extracts were loaded. To separate total protein extracts, equal amounts of protein were resolved by SDS-PAGE. Protein samples were subsequently blotted onto PVDF membranes. After blotting, membranes were blocked with Rotiblock (Roth) reagent and incubated with the respective primary antibody followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. HRP activity was detected using the SuperSignal West Femto Maximum Sensitivity kit (Thermo Scientific) and visualized by a LAS-4000 Mini bioimager (GE Healthcare Life Sciences). Signal intensities were quantified using Multi-Gauge software (GE Healthcare Life Sciences). Commercial antibodies used were HRP-conjugated α-HA (Roche), α-Histone H3 (Abcam), α-HSC70 (Stressgen), α-α-Tubulin (Sigma-Aldrich), α-rabbit IgG-HRP (Sigma-Aldrich) and α-mouse IgG-HRP (Sigma-Aldrich). α-SPA2 and α-COP1 antibodies were described previously in [42 (link)]. α-cry1 [67 ] and α-cry2 [68 (link)] antibodies were used to detect cry1 and cry2, respectively.