Mammalian lysis buffer

We cloned the Halo- or HA-tagged, full-length WT, R132H or Y208C mutant into the pFN21A (Promega) or pMACS Kk.HA-C vector, respectively. The expression of the Halo- and HA-tagged constructs in the HeLa cells was induced using FuGENE HD Transfection Reagent (Promega), and the transfected cells were grown for 48 h. The cells were then lysed and pulled down using Mammalian Lysis Buffer (Promega) containing Protease Inhibitor Cocktail (Promega) for 15 min, and cellular debris was cleared by centrifugation at 12,000 × g for 10 min. In total, 50 µl of Magne Halo-Tag Beads (Promega) equilibrated with TBS containing 0.05% IGEPAL CA-630 (TBS+) was added to the supernatant. The samples were incubated for 20 min at 22°C with rotation. The supernatant was discarded, and the protein-captured beads were washed thrice with TBS+ and suspended in SDS-PAGE loading buffer. The samples were analyzed by immunoblotting using anti-Halo or anti-HA antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare). The blots were developed using EzWestLumi plus reagents.

For the coimmunoprecipitation assay, wild-type or mutant myc-tagged ATP1A3 vectors and HaloTag-fused ATP1B1 vectors were cotransfected into HEK293T cells. At 48 hours after transfection, cells were lysed with Mammalian Lysis Buffer (Promega) containing Protease Inhibitor Cocktail (Promega) and PhosSTOP (Roche Diagnostics, Basel, Switzerland). The lysate was incubated with 2 μg of anti–Myc-tag mAb (MBL) at 4°C overnight and with Dynabeads Protein G (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C for 2 hours. After washing with phosphate-buffered saline (PBS), the beads were resuspended with 1% SDS buffer. Inputs and immunoprecipitates were subjected to Western blotting to detect ATP1A3 and ATP1B1 using anti–Myc-tag and anti-HaloTag antibodies. Because an excess of both myc-tagged ATP1A3 and HaloTag-fused ATP1B1 was confirmed in the flow-through after immunoprecipitation by Western blotting, and equivalent amounts of anti–Myc-tag mAb were added to the lysates for immunoprecipitation, we measured the band intensities of HaloTag signals from each immunoprecipitate. We then calculated the binding efficiency between mutant ATP1A3 and wild-type ATP1B1 as the HaloTag signal intensity of the particular immunoprecipitate divided by that of wild-type ATP1A3 and wild-type ATP1B1.

HeLa cells in 6-well plates were transfected with either WT, R132H or Y208C of cIDH1 expression plasmids (1 µg/well). After 48 h of transfection, the cells were lysed with mammalian lysis buffer (Promega) supplemented with a protease inhibitor cocktail (Promega). Post lysis, the samples were centrifuged (15,000 × g for 15 min at 4°C) to obtain the supernatant accordingly. Total protein levels were measured using BCA (Nacalai Tesque). Equal amounts of proteins (100 µg/condition) were then incubated with glutaraldehyde at different concentrations (0, 0.04, 0.1, 0.25 or 0.5%) and incubated on ice for 30 min accordingly. To make a working solution of glutaraldehyde, commercially available 25% glutaraldehyde solution was diluted in PBS and discarded after use. To quench the reaction, sample buffer was added to obtain the following final concentrations: 250 mM Tris-HCl, pH 8.5; 2% lithium dodecyl sulfate; 100 mM DTT; 0.4 mM EDTA; 10% glycerol; and 0.2 mM bromophenol blue. Samples were then separated by SDS-PAGE and the monomer and dimer of cIDH1 was detected using anti-HA antibody accordingly.

The cells were lysed in ice-cold Mammalian Lysis Buffer (Promega, Madison, WI, U.S.A.) containing Protease Inhibitor Cocktail (Promega) and incubated for 15 min at 4°C. Insoluble fragments
were removed by centrifugation at 16,000 × g for 10 min at 4°C, and the supernatant was collected. Protein concentrations were determined using the Bicinchoninate Protein Assay kit (Nacalai
Tesque). Further, cell lysates containing 10 µg of total protein were prepared for western blotting analysis by boiling samples in sodium dodecyl sulfate (SDS) sample buffer
(50 mM Tris, 2% SDS, 5% glycerol, 5% 2-mercaptoethanol, pH6.8).The prepared lysates were resolved on a 5–12.5% Tris-glycine polyacrylamide gradient gel using a Perfect Cell B from DRC
(Tokyo, Japan) (ΔV=150 V, 50 min at 25°C), transferred to a PVDF membrane using a standard semi-dry apparatus from Bio-Rad (Hercules, CA, U.S.A.) (ΔV=15 V, 1 hr at 25°C). After blocking with
10% skim milk, 6% glycine in TBS containing 0.1% Tween-20, the membrane was incubated with the following primary antibodies: mouse anti-ALDH1 (44/ALDH, 1:1,000, BD Biosciences, Franklin
Lakes, NJ, U.S.A.) and rabbit anti-β-actin (PM053, 1:2,000; MBL, Nagoya, Japan). Horseradish peroxidase-conjugated secondary antibodies and Ez WestLumi plus (ATTO, Tokyo, Japan) were used
for the detection of antibody-bound proteins.