Trans blot turbo mini 0.2um pvdf transfer packs membrane

The cells were washed with PBS and lysed with CelLytic M (C2978, Sigma-Aldrich, Saint Louis, MO, USA) lysis buffer containing protease inhibitors. The lysates were centrifuged at 12,000 rmp for 5 min, and the supernatant was collected. The proteins were separated by Mini-PROTEAN^® TGXTM Precast Gels (BioRad, Berkeley, CA, USA) and transferred to a Trans-Blot Turbo Mini 0.2um PVDF Transfer Packs membrane (BioRad, Berkeley, CA, USA) by using Trans-Blot^® TurboTM Transfer System (Bio-Rad, Berkeley, CA, USA). The antibodies for TOP2A (HPA006458), PLK1 (HPA053229), MCM2 (HPA031496, a-tubulin (ab7291, Abcam) and GAPDH (sc47724, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used for primary immunoblotting. All the antibodies were diluted at 1:1000 concentration. The membranes were incubated in primary antibody solution overnight at 4 °C with gentle rocking. Secondary antibody, goat Anti-Rabbit HRP (ab205718) or goat anti-mouse IgG-HRP (sc2005, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), was blotted for 30 min at 4 °C with gentle rocking. The protein bands were detected with ImageQuant LAS 500 (29-0050-63, GE) automatic exposure procedure or 10 min exposure.

Drug-induced glutaminase content was measured by Western blotting. The cells were washed with PBS and lysed with CelLytic M (C2978, Sigma-Aldrich, Saint Louis, MO, USA) lysis buffer containing protease inhibitors. The lysates were centrifuged at 12,000 rotations per minute for 5 min, and the supernatant was collected. Proteins were separated by Mini-PROTEAN^® TGXTM Precast Gels (BioRad, Berkeley, CA, USA) and transferred to a Trans-Blot Turbo Mini 0.2 um PVDF Transfer Packs membrane (BioRad, Berkeley, CA, USA) by using Trans-Blot^® TurboTM Transfer System (Bio-Rad, Berkeley, CA, USA). The antibodies for mitochondrial glutaminase-1 (kidney-type) and glutaminase-2 (liver-type) encoded from two GLS isomers and GAPDH were used for primary immunoblotting. All the antibodies were diluted at a 1:1000 concentration. DMSO was used as a blank reference. The membranes were incubated in primary antibody solution overnight at 4 ℃ with gentle rocking. The secondary antibody, goat Anti-Rabbit HRP (ab205718) or goat anti-mouse IgG-HRP (sc2005, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), was blotted for 30 min at 4 ℃ with gentle rocking. The protein bands were detected and band densities were quantified by Image J software.