Intelligent dark box

Protein samples were resolved using Novex 4–12% polyacrylamide gels with Tris glycine-SDS running buffer (Invitrogen, Grand Island, NY). After electrophoresis proteins from the gel were transferred onto a nitrocellulose membrane (Invitrogen, Grand Island, NY). The membranes were blocked in 5% non-fat milk/Tris buffered saline plus 0.1% Tween-20, (TBS-T) for 1 h at room temperature. Primary antibodies were goat anti-SGLT1 diluted at 1∶2,000 (Santa Cruz Biotechnology, Cat# sc-20582), goat anti-Glut2 diluted at 1∶2,000 (Santa Cruz Biotechnology, Cat# sc-31835), rabbit anti-GLUT1, diluted at 1∶500 (Abcam, Cat# ab32551), mouse anti β-actin, diluted 1∶2,000 (Thermo Scientific, Cat# 82353) in 5% non-fat milk/TBS-T. Secondary antibodies were HRP-conjugated donkey anti goat IgG, goat anti rabbit IgG, or goat anti-mouse IgG (KPL, Gaithersburg, MD), diluted at 1∶10,000 in 5% non-fat milk/TBS-T. For detection, membranes were incubated in SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Cat# 34080) and chemiluminescent bands were acquired using a Fuji Film Intelligent Dark Box with LAS-4000 software version 2.1.

For tissue protein extracts brain regions were snap-frozen in liquid nitrogen, grinded while frozen and lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4) supplemented with protease inhibitors (1 mM PMSF and Roche “complete mini” -tablets). Non-lysed debris was removed by centrifugation (25 min, 17000 g).
Equal amounts of protein (usually 20–50 μg) were seperated by SDS-PAGE under reduced-denaturing conditions. For an improved dissociation of glutamate transporter oligomers, samples were usually denatured with a twofold concentrated urea supplemented Laemmli loading buffer (200 mM Tris-HCl, 15% glycerol, 4% SDS, 6 M Urea, 5% β-mercaptoethanol). Proteins were transferred to PVDF membranes and blocked for 1 h with 5% dry milk in TBS buffer. Primary antibodies were incubated in the blocking solution overnight at 4 °C or for 2 h at room temperature, secondary antibodies for 1 h at room temperature. Luminescence signals were detected with the “Intelligent Dark Box” (Fuji). The membranes were usually reprobed several times with different antibodies, including anti-ERK2 as loading control.

A Western blot analysis was performed to probe for the Cpin_5740 gene product in native C. pinensis secretomes. As described above, total secretomes were collected from 50 mL liquid cultures of C. pinensis by centrifugation to pellet cells. Secretomes were concentrated approximately 50 times and utilised in Western blots. A total of 100 μg of each secretome was loaded and run on SDS-PAGE. TBST buffer (tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) with 0.1% Tween–20) was used for washing and dilutions throughout the Western blot protocol. The membrane was washed 5–6 times (~5 minutes each) between each step in the following procedure. After blotting proteins from an SDS-PAGE gel, the membrane was first blocked with a solution of TBST buffer + 3% BSA for 1 hour at room temperature to reduce non-specific binding. The purified antibodies were used as the primary antibody (1:1000 dilution, 1 hour incubation at room temperature with 1% BSA), with anti-rabbit IgG coupled to HRP (Sigma Aldrich) as the secondary antibody (1:10,000, 1 hour incubation at room temperature with 1% BSA). Final visualisation was achieved by chemiluminescence using the luminol-based Amersham ECL Western Blotting Detection Reagent (GE Healthcare) and a Fujifilm Intelligent Dark Box with LAS–1000 camera and software.