Femto ecl substrate

Astrocytes (95,000 cells/well, 24 well-plate) were lysed 48 h after treatment with 5× Laemmli buffer. Samples were incubated at 90 °C for 5 min, and proteins were separated on SDS-polyacrylamide gels. After electrophoresis, gels were transferred on to a PVDF membrane overnight at 40 V. Membranes were blocked with 5% non-fat milk (Sigma-Aldrich, St. Louis, MO, USA), followed by primary antibody incubation over night at 4 °C. Next, HRP-conjugated secondary antibodies were added (Agilent Dako, Santa Clara, CA, USA) to the membranes and kept for 1 h at RT. Finally, membranes were scanned with Femto ECL Substrate (Thermo Fisher Scientific, Waltham, MA, USA) using the Odyssey Fc system (LI-COR Bioscience, Lincoln, NE, USA). Images were quantified with Image Studio software (version 2.0.38). The following primary antibodies were used: rabbit anti-GFAP (1:1000; Agilent Dako, Santa Clara, CA, USA), mouse anti-Vimentin (1:200, Santa Cruz, Heidelberg, Germany), and mouse anti-actin (1:10,000, Sigma-Aldrich, St. Louis, MO, USA). Total protein levels for GFAP and Vimentin were normalized to actin.

Yeast cultures were grown in YP+2% galactose or YP+2% dextrose to express or suppress TAP–Tom5, respectively. Log phase cultures (30 OD₆₀₀ units) were collected by centrifugation and resuspended in 400 µl of lysis buffer (250 mM NaCl, 50 mM Tris-HCl pH 7.4, 50 mM NaF, 5 mM EDTA, 1 mM DTT, 1 mM AEBSF, and 0.1% NP-40). Cells were disrupted with glass beads for 5 min at 4°C. The resulting lysate was centrifuged at 16,100×g for 5 min, and a sample of the supernatant was collected. The supernatant was then incubated under rotation with 20 µl of prewashed GFP–Trap_A bead slurry (Chromotek) for 1 h at 4°C. The supernatant was removed and the beads were washed with lysis buffer (3×1 ml, then 3×500 µl with 5-min incubations). Beads were resuspended in 100 µl of sample buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 4% 2-mercaptoethanol, and 0.02% bromophenol blue) and eluted by heating at 65°C for 20 min. The eluted protein was subjected to SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-TAP (1∶5,000 dilution, Pierce, CAB1001) or anti-GFP (1∶5,000 dilution, Roche, 11814460001) antibodies. Goat anti-rabbit IgG-HRP (1∶5,000 dilution, Bio-Rad, 172-1019) and goat anti-mouse IgG-HRP (1∶5,000 dilution, Bio-Rad, 172-1011) were used as secondary antibodies. Blots were imaged with either Supersignal West Pico or Femto ECL substrate (Thermo Scientific).

Synaptosome samples were mixed with 5× Laemmli buffer and incubated at 90 °C for 5 min. Proteins were separated on SDS-polyacyrlamide gels containing 2,2,2-trichloroethanol for total protein amount visualization. After electrophoresis, gels were scanned using a Gel Doc EZ imager (Bio-Rad) and electro-transferred onto a PVDF membrane overnight at 40 V. Membranes were blocked with 5% non-fat milk (Sigma-Aldrich, St. Louis, MO, USA), incubated with primary antibody at 4 °C for 1 h and then with matching HRP-conjugated secondary antibodies at 4 °C for 1 h (Agilent Dako, Santa Clara, CA, USA). After washing, the membranes were scanned with Femto ECL Substrate (Thermo Fisher Scientific, Waltham, MA, USA) using the Odyssey Fc system (LI-COR Bioscience, Lincoln, NA, USA). Images were quantified using Image Studio software (version 2.0.38). Differences in loading were corrected using the quantification of the total protein amount and immunoblot signals were normalized to the controls. The following primary antibodies were used: anti-ME3 (Abcam, Cambridge, UK; ab172972), anti-mGluR5 (GenScript, Piscataway, NJ, USA; A01493), anti-Rat mGluR1α (BD Biosciences, San Jose, CA, USA; 556331) and anti-PSD-95 (NeuroMab, Davis, CA, USA; 75-028).