Anti gls1

Proteins were incubated with anti-USP6 (Bethy Laboratories, A305-225), anti-GLS1 (Cell Signaling Technology, #56750), or control IgG (Beyotime) for 1 h, followed by incubation with protein A/G-agarose for 3 h at 4 °C. Precipitates were washed and submitted for Western blot analysis.

Cell pellets were lysed in the lysis buffer [10 (link)] added with protease and phosphatase inhibitor cocktail for 30 min on ice. Insoluble material was pelleted at 10,000× g for 15 min at 4 °C, and the protein concentration was determined using a BioRad assay kit (BioRad, Hercules, CA, USA). Thirty μg of total cellular proteins were separated on SDS-PAGE and electrotransferred to activated PVDF membrane (Merck Millipore, Burlington, MA, USA). Immuno-blotting was carried out with anti-GLS1 (Cell signaling, 1:1000), anti-GPT2 (Santa Cruz Biotechnology, Dallas, TX, USA 1:250) and anti-RAN (Santa Cruz Biotechnology, Dallas, TX, USA 1:500) primary antibodies and anti-mouse and anti-rabbit peroxidase labelled secondary antibodies (BioRad, Hercules, CA, USA). Horseradish-peroxidase substrate (ECL Western Blotting Detection, Amersham-Life Science, Little Chalfont, UK) was added and the signal was revealed through an Odyssey Fc instrument (LI-COR, Lincoln, NE, USA).