Adi sab 100 j

Lysate extracted from a total of 1 × 10⁵ cells was used to perform Western blots analysis. Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used. Western blotting was followed by incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase (HRP), anti-rabbit IgG-HRP (1:5000; Enzo, Farmingdale, NY, USA, ADI-SAB-300-J), and anti-mouse IgG-HRP (1:5000; Enzo, ADI-SAB-100-J). The immunoreactive bands were developed with the chemiluminescence ECL Detection System (GE Healthcare, Chicago, IL, USA), and signals were detected using a luminescent image analyzer (LAS-4000 Mini, Fujifilm, JPN).

SDS–PAGE was performed by loading extracted protein samples on an 8–12% gel with a protein marker (PM2610, SMOBiO, Hsinchu City, Taiwan, R.O.C.). The separated proteins on the gel were electrotransferred to a PVDF membrane (AE-6667-P, Atto, Taito-ku, Tokyo, Japan) at 350 mA for 70 min. The membranes were blocked with 5% skim milk (232100, BD Difco, USA) in PBST and incubated with primary antibodies in 1% skim milk overnight at 4 °C. The next day, the membranes were incubated with HRP-conjugated secondary antibodies (ADI-SAB-300-J, goat anti-rabbit IgG (HRP conjugate) and ADI-SAB-100-J, goat anti-mouse IgG F (ab’)2 (HRP conjugate); Enzo Life Sciences, Farmingdale, NY, USA) in 2% skim milk for more than an hour at RT, and chemiluminescent signals were then detected using EzWestLumi plus reagent (2332637, Atto), a LAS3000 imager (Fujifilm) and an Amersham™ ImageQuant™ 800 biomolecular imager. The antibodies used to identify proteins during western blotting are described in Supplementary Table 1.

An amount of 30 μg of total protein per lane were loaded on 10% bis-acrylamide gels, and electrophoresis was performed at 80 volts for 2 h. After being transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden), the membranes were blocked in 5% skim milk for 1 h under agitation and probed with human α -tubulin (diluted 1:10,000), ICAM-1 (diluted 1:1000), ERK (diluted 1:5000), phospho-ERK (diluted 1:2000), JNK (diluted 1:5000), phospho-JNK (diluted 1:2000), p38MAPK (diluted 1:5000), phospho-p38MAPK (diluted 1:2000), AKT (diluted 1:5000), and phospho-AKT (diluted 1:2000) antibodies for 16 h at 4 °C. After incubation with primary antibodies, the membranes were washed three times for 10 min with phosphate-buffered saline (PBS) and incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat-anti-rabbit IgG (diluted 1:10,000, #ADI-SAB-500-J, ENZO Life Science, Farmingdale, MY, USA) or goat-anti-mouse IgG (diluted 1:10,000, #ADI-SAB-100-J, ENZO Life Science) as the secondary antibodies. The signal was developed using an enhanced chemiluminescence solution (Amersham Pharmacia Biotech) and visualized on an Azure 300 Imaging system (Azure Biosystems, Dublin, CA, USA).

The information about chemicals and antibodies used in the method is as follows: charged aerosol detector (CAD) (Thermo Scientific, Waltham, MA, USA), 0.1% fomic acid (Sigma Aldrich, Saint Louis, MO, USA), acetonitrile (Sigma Aldrich), amyloid-β (Santa Cruz, Dallas, TX, USA, sc-53822, 1:500), BACE1 (Invitrogen, Waltham, MA, USA, PA5-19952, 10 μg/mL), APP (Invitrogen, 14-9749-82, 2.5 μg/mL), Iba-1 (Abcam, Cambridge, UK, ab178846, 1:2000), GFAP (BD Biosciences, Dickinson, ND, USA, BD-556328, 5 μg/mL), β-actin (Santa Cruz, sc-8432, 1:1000), phospho-Tau (Invitrogen, 44-752G, 1:1000), BACE1 (Invitrogen, PA5-19952, 1:1000), phospho-JNK (Santa Cruz, sc-6254, 1:1000), phospho-ERK (Santa Cruz, sc-7383, 1:1000), phospho-p38 (Cell Signaling, Danvers, MA, USA, csD3F9, 1:1000), anti-goat mouse (Enzo Life Sciences, Farmingdale, NY, USA, ADI-SAB-100-J, 1:2000), and anti-goat rabbit (Enzo Life Sciences, ADI-SAB-300-J, 1:2000).

FLAG M2 beads were blocked with 3% bovine serum albumin in PBS for 1 h and equilibrated in the reaction buffer (tSETD2-FLAG gel filtration buffer: 50 mM NaPi, pH 7.2, 150 mM NaCl, 5 mM β-mercaptoethanol, 5% glycerol). tSETD2-FLAG protein (WT or variants) was added at 20 μM with a putative binding partner for 2 h in the presence of SAM. For tubulin, 0.25 mg/ml of porcine tubulin (Cytoskeleton, Inc.) was used, and for RNA Pol II, 10 μl of HEK293 FreeStyle clarified lysate was used. Beads were spun down, and the supernatant was collected as the fraction of unbound substrate. The beads were then resuspended in the reaction buffer to the total reaction volume, and the same amount of supernatant and beads was added to SDS-PAGE gel. Analysis of binding was conducted by Western blot with the following antibodies: anti-FLAG (1:1000, A9469; Sigma Aldrich), anti-tubulin E7 (1:1000, AB_528499; DSHB) and/or TU-01 (1:1000, 625902; BioLegend), and anti-RNA Pol II (1:1000, ab193468; Abcam), with secondary antibody anti-mouse (1:1000, ADI-SAB-100-J; Enzo Life Science) or anti-rabbit (1:1000, ADI-SAB-300-J; Enzo Life Science), respectively. Binding was quantified by measuring the background-subtracted intensity of each band with Fiji ImageJ (65 (link)) as a fraction of the input intensity. Each experiment was performed three times, independently.