Mouse anti gfp

HEK293T and SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) with 10% FBS (Gibco), and 1% penicillin/streptomycin (Gibco) at 37°C in a 5% CO₂ incubator. Lipofectamine 2000 (Invitrogen) was used for plasmid transfection. The components of cell lysis buffer were 1% NP-40, 150 mM NaCl, 1 mM NaF, 50 mM Tris, pH 7.6, and protease inhibitor mixture (Roche). Cells were lysed in lysis buffer at 4°C for 1 h. After centrifugation at 4°C at 12,000 g for 10 min, the supernatants were collected and incubated with the primary antibody at 4°C for 3.5 h. The immune complexes were associated with protein A/G agarose beads (Santa Cruz) at 4°C for 2.5 h. The beads were then washed six times and boiled for 5 min. The Western blot analyses of UNC-103 protein levels were not conducted blind to genotypes, but two researchers independently did the experiments and obtained similar results. The following antibodies were used in these experiments: rabbit anti-hERG (Millipore), rabbit anti–c-Myc (Sigma), rat anti-HA (Roche), mouse anti-tubulin (Sigma), mouse anti-GFP (Abmart), and rabbit anti-actin (Abmart).

For Western blot analysis, cells were harvested and lysed in RIPA lysis buffer (Cell Signal Technology). To investigate subcellular distribution of proteins, nuclear and cytoplasmic fractions were enriched using Nuclear and Cytoplasmic Protein Extraction Kit (CoWin Bioscience). Western blot assays were followed according to an established protocol (20 (link)). Antibody used included mouse anti-GFP (Abmart), rabbit anti-acetyl Lysine (Abcam), rabbit anti-SUMO1 (Abcam), rabbit anti-Lamin B1 (Abcam), mouse anti-Ub (Santa Cruz), rabbit anti-USP49 (NOVUS), mouse anti-Flag (Proteintech), mouse anti-GAPDH (Thermo Fisher Scientific), mouse anti–β-tubulin (Thermo Fisher Scientific), rabbit anti-GRα (Abcam), rabbit anti-GRβ (Abcam). For protein stability detections, cells were treated with proteasome inhibitor MG132 (20 μmol/L, Sigma-Aldrich) for 6 hours or protein synthesis inhibitor Cycloheximide (CHX, 100 μg/mL, Sigma-Aldrich) for indicate time before harvest.