Tubulin

Cells were fixed by 4% paraformaldehyde (Sigma Aldrich) for 15 min, permeabilized by 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, blocked by 3% bovine serum albumin (BSA, Sigma-Aldrich) for 20 min, and stained by anti-TUBB3 (neuronal class III β-tubulin) (1:1000; Covance, Princeton, NJ, USA) antibody at 4° C overnight. The cells were washed by phosphate-buffered saline (PBS) for twice and stained with the secondary Alexa Fluor ®555 goat anti-rabbit antibody (1:1000; Molecular probes) at room temperature for 3 h, and with 4’-6-diamidino-2-phenylindole (DAPI, 0.1 μg/ml, Sigma-Aldrich) for 30 min. Images of neurites were captured by Micro Confocal High-Content Imaging System (Molecular Devices), and analyzed by MetaXpress Neurite Ougrowth Application Module (Molecular Devices).

Cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Cell Signaling Technology; cat # 5781). Protein lysates were subjected to SDS/PAGE and immunoblotted with antibodies against the MED19 isoforms or MYC tag (Cell Signaling Technology; cat # 2276S). Tubulin (Covance; cat # MMS-489P) was used as a loading control. Protein bands were visualized using a Clarity Western ECL Substrate (BioRad; cat #1705060), and images were acquired on an iBright CL1000 (ThermoFisher Scientific) using the “Auto-Exposure” option in the iBright Imaging System software. This feature utilizes software algorithms to analyze all signals present within a selected frame automatically. Imaging parameters are as follows: Zoom level = 1.2×; Focus level = 246; Resolution = 5 × 5; Exposure mode = normal; Exposure times vary depending on the antibody and amount of immunogen: 0.3–10 s.