Proteinase inhibitors

Human samples were homogenized in RIPA lysis buffer with proteinase inhibitors (Santa Cruz Biotechnology, Dallas, TX, United States, sc24948). Protein extracts, 40 μg per lane, were loaded onto 4–20% gradient gels (NuSep Inc., Germantown, MD, United States, NB10-420). Gels were electrotransferred to a 0.2 μm nitrocellulose membrane (Bio-Rad, 1620174). Blots were blocked in 5% milk in tris-buffered saline, 0.1% Tween 20 (TBST) for 1 h, and then incubated at 4 °C overnight with one of the following antibodies raised against: AATM (1:1000, sc-271702, Santa Cruz Biotechnology), AQP4 (1:1000, sc-390488), CX30 (1:1000, sc-81802), CX43 (1:1000, sc-271837), EAAT1 (1:500, sc-515839), GABRA1-6 (1:250, sc-376282), GAPDH (1:2000, sc-32233), GAT-3 (1:250, sc-376001), GDH1/2 (1:1000, sc-515542), GEPH (1:1000, sc-25311), GS (1:1000, sc-74430), MAOB (1:250, sc-515354), SNAT5 (1:1000, sc-515813), VIAAT (1:500, sc-393373), GBRD (1:250, Novus Biologicals, Littleton, CO, United States, NB300-200) or GABRG1 (1:100, Alomone Labs, Jerusalem, Israel, AGA-016). Bands were detected with appropriate horseradish peroxide-conjugated secondary antibodies, reacted with chemiluminescent ECL substrate (Bio-Rad, 1705060) and visualized with a Bio-Rad ChemiDoc Imaging system. Band intensity was measured using the ImageJ program (NIH) and normalized with GAPDH.

Human samples were homogenized in RIPA lysis buffer with proteinase inhibitors (Santa Cruz Biotechnology, sc24948). Protein extracts, 40 μg per lane, were loaded onto 4–20% gradient gels (NuSep Inc, NB10-420). Gels were electrotransfered to a 0.2-μm nitrocellulose membrane (Bio-Rad, 1620174). Blots were blocked in 5% milk in TBST for 1 h, and then incubated at 4°C overnight with one of the following antibodies: anti-gad65; anti-gad67; anti-SST; anti-gapdh (Santa Cruz Biotechnology, sc377145, sc28376, sc7819, and sc32233, respectively), anti-CLB (Swant Marly, Cb38), and anti-CRT (Millipore Sigma, AB1550). Bands were detected with appropriate horseradish peroxide-conjugated secondary antibodies, reacted with chemiluminescent ECL substrate (Bio-Rad, 1705060) and visualized with a Bio-Rad ChemiDoc Imaging system. Band intensity was measured using the ImageJ program (NIH).

Cells were infected with Ad-NDRG4 or control virus (10 MOI) for 72 h. Then, cells were collected and lysed using RIPA (Radioimmunoprecipitation assay) buffer with supplements of proteinase inhibitors and phosphatase inhibitors (Santa Cruz, CA, USA). Western blotting was carried out following standard protocol. The antibody against NDRG4 was purchased from LSBio (LS-C133806, mouse monoclonal, Seattle, WA, USA). Antibodies against CyclinD1 (E3P5S, monoclonal), CDK4 (D9G3E, monoclonal), and CDK6 (DCS83, monoclonal) were purchased from Cell Signaling (Danvers, MA, USA). β-Actin (AC-74, Millipore Sigma, Miamisburg, OH, USA) was used as internal loading control of each experiment.