Ecl chemostar

50 mL yeast cultures in S.D. media were harvested and disrupted using acid-washed glass beads [23 (link)]. The cell lysates were separated by SDS-PAGE and proteins were transferred to polyvinylidene fluoride membranes (Amersham Hybond-P 0.45, GE Healthcare Life Sciences, München, Germany). Chemiluminescence detection (ECL Chemostar, Intas Science Imaging Instruments, Göttingen, Germany) was carried out using a monoclonal mouse Anti-HA antibody (1:5000, Roche; cat. no. 11583816001, Mannheim, Germany), a horseradish peroxidase secondary goat anti-mouse antibody (1:5000, Jackson ImmunoResearch/Dianova; cat. no. 115-035-174, Hamburg, Germany), and the Clarity Western ECL substrate (Bio-Rad, Feldkirchen, Germany).

MARCKS protein levels in whole cell extracts of WT, IM, and KO cells as well as PKCβ levels in PKCβ KO cells were initially detected using the Western Blot technique as previously described [33 (link)]. For protein detection, membranes were incubated (4 °C, overnight) with primary antibodies specific for MARCKS (D88D11 XP^®), phosphorylated (p)-MARCKS (Ser167/170; D13E4 XP^®), PKCβ (D3E70), Akt (C67E7), or p-Akt (Thr308; D25E6 XP^®; Cell Signaling Technology, Danvers, MA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma Aldrich, St. Louis, MO, USA). Following incubation with horseradish peroxidase (HRP)-coupled secondary antibodies (Vector Laboratories/Alexis, Grünberg, Germany), protein bands were visualized using the detection reagents enhanced chemiluminescence (ECL; Thermo Fisher, Bonn, Germany) or WesternBright Sirius (Advansta, Menlo Park, CA, USA) and the Bio-imaging system ECL Chemostar (Intas Science Imaging, Göttingen, Germany).

Preparation of whole-cell, cytosolic, and nuclear extracts, determination of protein concentrations, electrophoresis, and Western blotting were performed as previously described [7 (link)]. For the detection of specific proteins, membranes were incubated (4 °C, overnight) with primary antibodies. Following incubation with horseradish peroxidase-coupled secondary antibodies, protein bands were visualised using enhanced chemoluminescence (ECL), SuperSignal West Femto (Thermo Fisher), or WesternBright Sirius (Advansta, Menlo Park, CA, USA) and the Bio-imaging system ECL Chemostar (Intas Science Imaging, Göttingen, Germany). For densitometric analyses, the ImageJ analysis software (National Institutes of Health, Bethesda, MD, USA) was used.

Gels were prepared with 1.5 weight (wt) % agarose in TBE buffer (22.25 mM tris base, 22.25 mM boric acid, 0.5 mM EDTA, and 6 mM magnesium acetate) and cast without stain. Gels were run at 3 V/cm in a water-cooled CBS Scientific HSU-020 gel electrophoresis chamber set to 15°C for 2.5 hours using an Enduro 300 V power source. Gels were imaged once with light-emitting diodes and filters set for Cy3 and Cy5, then poststained in 1× SYBR, and imaged again using an INTAS ECL ChemoStar.