Anti gapdh

Neuroblastoma cell lines were seeded in a 10-cm dish at a final density of 1.5×10⁶ cells and allowed to attach overnight. The cells were then treated with DMSO or experimental compounds. After 48–72 h, cells were lysed in CHAPS lysis buffer with protease and phosphatase inhibitors (Roche, Basel, Switzerland), and 25 or 50 μg of total lysate were boiled for 5–10 min in CHAPS and sample buffer. Western blotting was performed using the following primary antibodies: anti-ALK (Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-ALK (Cell Signaling Technology), anti-Shc (Cell Signaling Technology), anti-Phospho-Shc (Cell Signaling Technology), anti-PARP (Cell Signaling Technology), anti-cleaved Caspase-3 (Cell Signaling Technology), anti-GAPDH (Fuji Film-Wako Chemicals), and anti-Actin (Merck). Secondary antibodies used were as follows: HRP-linked anti-rabbit IgG (Cell Signaling Technology) and HRP-linked anti-mouse IgG (Cell Signaling Technology). The proteins were visualized using ImageQuant LAS 4000mini with enhanced chemiluminescence reagents (Thermo Fisher Scientific).

KR12‐treated cells were lysed using RIPA buffer containing phosphatase and complete proteinase inhibitor. BCA protein assay kit (Thermo Fisher Scientific) was used to determine protein concentration. Proteins were separated by SDS‐PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 60 min at room temperature and subjected to immunoblotting using the primary antibodies of anti‐KRAS (sc‐30; Santa Cruz Biotechnology), anti‐RAD51 (ab133534; abcam) and anti‐GAPDH (016–25,523; Wako). The results were quantified using the ImageJ software.

Cells lysed on ice in lysis buffer [20 mM Tris-HCl (pH 7.4), 135 mM NaCl, 1% Triton-X 100, 10% glycerol] supplemented with a protease inhibitor cocktail, cOmplete mini (MilliporeSigma), were boiled in loading buffer and subjected to 5%–20% gradient SDS-PAGE. The proteins were transferred to polyvinylidene difluoride membranes (MilliporeSigma) and incubated with the anti-SARAS-CoV-2 S antibody (1:5,000 dilution; GeneTex), anti-N antibody (1:5,000 dilution; Sino Biological), or anti-GAPDH (1:5000 dilution; FUJIFILM Wako). The immune complexes were visualized with SuperSignal West Femto substrate (Thermo Fisher Scientific). The signals were detected using a WSE-LuminoGraph I (ATTO) and ImageSaver6 (ATTO).

The expression vectors of HA-tagged CPT1 or HA-tagged CEPT1 described previously were transfected into DKO cells using Lipofectamine 2000. After 24 h, the cells were lysed in 20 mM Tris-HCl buffer, pH 8.0, containing 0.2% Triton X-100 (w/v) and proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Proteins were separated with SDS-PAGE, transferred to PVDF membranes (FluoroTrans; Pall Corp., Port Washington, NY) using a Trans-Blot SD Semi-Dry Transfer blotter (Bio-Rad Laboratories, Hercules, CA), and then the membranes were incubated with 5% (w/v) skim milk in TBS for 1 h. The membranes were then incubated with anti-HA tag (Cell Signaling Technology) and anti-GAPDH (Wako Pure Chemical Industries) antibodies overnight at 4°C, washed three times with TBS containing 0.1% Tween-20 (w/v), and then incubated with horseradish peroxidase-conjugated IgGs for 1 h at room temperature. The membranes were washed three times with TBS containing 0.1% Tween-20 and stained with Clarity Western ECL Substrate (Bio-Rad) according to the manufacturer's instructions and visualized using a ChemiDoc Touch imaging system (Bio-Rad). Protein density was quantified using Image Lab software (Bio-Rad). Results were normalized against the density of GAPDH.

The cells were harvested in a 2% sodium dodecyl sulfate (SDS) buffer. Lysates were then incubated at 95 °C for 5 min. The protein concentration of each lysate was examined using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Whole cell lysates (approximately 15 µg) were separated using SDS-polyacrylamide gel electrophoresis and separated proteins were then transferred to polyvinylidene fluoride membranes (Immobilon P; EMD Millipore, Burlington, MA, USA). The membrane was blocked for 1 h in 5% skimmed milk (Fujifilm-Wako, Osaka, Japan). Immunoblotting was conducted using anti-OSGIN1 (1:1000 dilution; 15248-1-AP, Proteintech, Chicago, IL, USA), anti-NRF2 (1:1000 dilution; 16396-1-AP, Proteintech), anti-cleaved caspase-3 (1:1000 dilution; 9661, Cell Signaling Technologies, Danvers, MA, USA), and anti-GAPDH (1:5000 dilution; 015-25473, Fujifilm-Wako). Horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000 dilution; 458, Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) was used as the secondary antibody. Primary antibodies and secondary antibodies were diluted with Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo, Osaka, Japan) and Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo), respectively. Protein band images were acquired using a ChemiDoc Touch Imaging System (Bio-Rad) and analyzed using Image Lab Software (version 5.2.1, Bio-Rad).

Cells were lysed with radioimmunoprecipitation assay buffer (Fujifilm Wako Pure Chemical) containing protease/phosphatase inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were transferred onto polyvinylidene difluoride membranes. After blocking, the blots were incubated with the following antibodies: anti‐p21^Cip1/Waf1 (#2947; Cell Signaling Technology [CST]), anti‐p16^Ink4a (SPC‐1280; StressMarq Biosciences), anti‐γH2AX(Ser¹³⁹) (#9718; CST), anti‐Bcl‐2 (#658701; BioLegend), anti‐Bcl‐xL (#2764; CST), anti‐Mcl‐1 (#54535; CST), anti‐survivin (#71G4B7; CST), anti‐cFLIP (ALX‐804–428; Enzo Life Sciences), anti‐TATA‐binding protein (TBP; #22006‐I‐AP; Proteintech), anti‐PARP (#46D11; CST), anti‐caspase‐3 (#9668; CST), anti‐c‐Myc (1472–1; EPT), anti‐GAPDH (#015‐25473; Fujifilm Wako Pure Chemical), and anti‐β‐actin (#622102; BioLegend). Nuclear and cytoplasmic proteins were prepared using the LysoPure™ Nuclear and Cytoplasmic Extraction Kit (Fujifilm Wako Pure Chemical). After washing, membranes were incubated with goat anti‐rabbit or horse anti‐mouse horseradish peroxidase‐conjugated secondary antibody (#7074 and #7076; CST). Protein bands were visualized using an Amersham ImageQuant™ 800 Biomolecular Imager (General Electric Company).