Mini protean tgxtm precast gel

Samples containing 50 µg protein of lysed spheroids (day 6) and cells grown in monolayer were separated on Mini-PROTEAN® TGX^TM Precast gels (Bio-rad, United States) as described previously [19 (link)]. Antibodies used for target detection; LDH-A (47010, 1:1000, Abcam, United Kingdom), β-actin (4790, 1:1000, Cell signalling, United States), Secondary IRDye® 800CW (926-32230, 1:10 000, Li-COR Bioscience, United States).

For protein isolation cells were cultured in 6-well plates; at the end of the experiment, cells were briefly washed in ice-cold PBS followed by incubation with 300 µL ice-cold RIPA buffer (Sigma Aldrich) containing a cocktail of protease inhibitors (Roche). The cells were scraped from the culture surface and incubated on ice for 15 min; then, they were homogenized through a 21 G needle. Samples were centrifuged at 8000× g for 15 min at 4 °C and the supernatant was transferred to a fresh tube and frozen for later quantification and use. Proteins were resolved using Mini-PROTEAN TGX^TM Precast gels (Biorad, Watford, UK) and transferred to nitrocellulose membranes using the TransBlot Turbo system (Biorad). Membranes were incubated in primary antibodies overnight (PC2, Santa Cruz Biotechnology, Sc-25749), beta-actin (Abcam, Ab8226) and immunoreactive bands were labeled using LI-COR near-infrared secondary antibodies and quantified using Li-Cor Image Studio™ Lite.

Proteins were extracted from cell lines using mammalian protein extraction reagent (MPER; 50 nM Tris-HCl, 200 mM NaCl, 0.25% Triton 100X, and 10% glycerol) containing a protease and phosphatase inhibitor cocktail (ThermoFisher, Waltham, MA, USA; PIA32961). Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA; 500–0006). Twenty micrograms of protein were separated in precast 4–15% gradient Tris-glycine SDS-polyacrylamide gels (Mini-PROTEAN^® TGXTM Precast Gels, Bio-Rad, Hercules, CA, USA; 456–1086) and transferred in Trans-Blot Turbo Mini 0.2 μm nitrocellulose membranes (Bio-Rad, Hercules, CA, USA; 170–4159). Membranes were blocked with 5% milk in PBS-Tween for 1 h and incubated overnight at 4 °C with primary antibody in BSA 1%. Membranes were next incubated with secondary antibody for 1 h, and signal was detected using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). ß-Actin was used as a loading protein control and each experiment was repeated three times. Image J [20 (link)] was used to quantify protein levels in Western blots.

Total proteins were isolated from cells incubated with RTV, LPV or DMSO using Lysis Buffer (NP40 Cell Lysis Buffer; Invitrogen^TM, Carlsbad, CA, USA) combined with a protease inhibitor cocktail 100× (1× final; Protease Inhibitor Cocktail Set I, Calbiochem^®, EMD Chemicals Inc., Merck KGaA, Darmstadt, Germany) and a phosphatase inhibitor cocktail 50× (1× final; Phosphatase Inhibitor Cocktail 50× Set V, Calbiochem^®, EMD Chemicals Inc., Merck KGaA, Darmstadt, Germany). Proteins (20 µg) were loaded on 4–15% gradient gel (Mini-PROTEAN^® TGX^TM Precast Gels, BIORAD^®, Hercules, CA, USA), and were then transferred on nitrocellulose membrane which was blocked with TBS 1×-Milk 5%-Tween 0.1% solution. The resulting proteins blots were probed with anti-MLN64, anti-P450SCC, anti-HSD3B1, anti-OPA1, anti-Mfn2, anti-GRP78, monoclonal mouse anti-actine or anti-vinculine antibodies (references and concentration in Table 1) [11 (link),17 (link)]. Actine or vinculine were used as loading control. Addition of secondary goat anti-mouse antibody conjugated with DyLight 680 (1/15,000, #35518 Thermo Fisher Scientific, Waltham, MA, USA) or secondary goat anti-rabbit antibody conjugated with DyLight800 4× PEG (1/15,000, SA5-35571 Thermo Fischer Scientific, Waltham, MA, USA) allowed blots revelation using Odyssey infrared fluorescent system (LI-COR).

Western blots were performed with Mini-PROTEAN^® TGX^TM Precast Gels from Bio-Rad (California 94547 United States) gradient 4–15% Gels were transferred onto Nitrocellulose blotting membrane (GE Healthcare Life Sciences) using Towbin buffer (25 mM Tris, 192 mM Glycine, 20% Methanol). Membranes were blocked with Tris–buffered saline TBS (50 mM Tris ph7–150 mM NaCl) with 5% Milk for 1 h at room temperature, incubated with primary and secondary antibodies as indicated below, and then developed with Bio-Rad’s Clarity ECL on ChemiDoc Touch Imaging System (Biorad). For Western blot, 30 ug of protein extract were used.

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.