Can get signal

Immunoblot analysis was performed as previously reported^{2 (link),25 (link)}. Isolated glomeruli were lysed by lysis buffer composed of 150 mM NaCl, 3 mM KCl, 5 mM EDTA, 3 mM ethylene glycol tetraacetic acid (EGTA) and 50 mM Tris-HCl (pH 7.4) containing 1% Triton X-100. After centrifugation at 3,000 × g at 4 °C for 10 min to remove cell debris and magnetic beads, the supernatants were carefully collected. 5–10 μg of protein was loaded into each lane for Laemmli SDS-polyacrylamide gel electrophoresis (8–12.5%), and then transferred to a polyvinyliden difluoride membrane. The membrane was blocked for 1 h using 2.5% milk powder in TBST (10 mM Tris-HCl, pH 8.5, 150 mM NaCl and 0.1% Tween 20), and exposed to primary antibodies diluted with Solution 1 (Can Get Signal; TOYOBO, Osaka, Japan) overnight at 4 °C. After a TBST rinse, the secondary antibody diluted with Solution 2 (Can Get Signal; TOYOBO, Osaka, Japan) was applied to the membrane for 1 h at room temperature. After washing, antigen–antibody complexes were visualised with a chemiluminescence system (Immobilon, Merck Millipore, Darmstadt, Germany).

Culture broth in CD liquid medium incubated for 24 h was filtered through Miracloth. Proteins in the supernatant were precipitated with trichloroacetic acid, separated by SDS-PAGE, and then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with the polyvinylidene difluoride blocking reagent for Can Get Signal (Toyobo). Antibodies of rabbit IgG against AmyD were developed with synthesized peptides (NH₂-C+SGERAGELDVPMSK-COOH) (Eurofins Genomics, Tokyo, Japan) and used as the primary antibody, diluted with Can Get Signal (Toyobo). Antibody binding was visualized using a horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and an ImmunoStar LD chemiluminescent substrate (Fujifilm Wako Pure Chemical Corp., Osaka, Japan).

Protein extracts were prepared essentially as described previously [40 (link)]. Briefly, cells grown to exponential phase were incubated with YPD or SD medium containing 2 μg/ml tunicamycin, 4 mM DTT or 0.4 M sodium chloride, for the indicated times. Cells were transferred into test tubes, mixed 1:1 with boiled medium, submerged in the boiling water for 3 min, and harvested by centrifugation. Cells were then subjected to a mild alkali treatment-based protein extraction method [41 (link)]. Western blot analysis was performed using the immunoreaction enhancer solution Can Get Signal (Toyobo) according to the manufacturer's protocol. Anti-HA monoclonal antibody 16B12 (Covance), anti-Myc monoclonal antibody 9E10 (Santa Cruz), anti-GFP monoclonal antibody JL-8 (Clontech), anti-phospho-p38 MAPK monoclonal antibody 28B10 (Cell Signaling), anti-phospho-AMPKα monoclonal antibody 40H9 (Cell Signaling), anti-Hog1 polyclonal antibody y-215 (Santa Cruz), anti-Snf1 polyclonal antibody yk-16 (Santa Cruz), and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Westren (Merck Millipore). Signal intensities were quantified by ImageQuant (GE Healthcare), and statistical analysis was performed with Excel (Microsoft).

U87 cells were incubated in each condition for 72 h in 5% CO₂ at 37°C. Immunofluorescence was carried out according to established techniques.³⁰ (link) U87 cells were fixed with 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque) for 10 min and permeabilized in 0.2% Triton X-100/PBS for 5 min. Samples were blocked with Blocking One (Nacalai Tesque), and the primary antibody was diluted with Can Get Signal® (Toyobo).

For immunological detection, 20 μg of cell lysate was separated using SDS-PAGE (7.5–10% Tris gel). Then, the proteins were blotted onto a PVDF membrane, which was incubated with the PVDF blocking reagent Can Get Signal (TOYOBO, Osaka, Japan). Proteins were probed with primary antibodies against β-catenin (1/1000, Abcam, Cambridge, MA, USA), FOXF2 (1/1000, Abnova, Walnut, CA, USA), WNT2B (1/2000, Abcam), LMNB1 (1/2000, Proteintech, Rosemont, IL, USA), and GAPDH (1/2000, MBL, Aichi, Japan). A horseradish peroxidase-conjugated goat anti-rabbit antibody was then added, and secondary antibodies were detected through autoradiography using enhanced chemiluminescence (ECL Plus, General Electric Healthcare, Chicago, IL, USA).

Plasmids (2 μg) containing WT METTL23-FLAG, splicing 1–FLAG (skip exon 2), and splicing 2–FLAG (skip exons 2 and 3) were transfected into subconfluent HEK293T, COS-7 (ATCC), and 661W cells (62 (link)) in a 6-well plate using ViaFect transfection reagent (Promega), respectively. Forty-eight hours later, the cells were harvested by RIPA buffer with protease and phosphatase inhibitors (Roche), PMSF, and aprotinin. Equal amounts of samples were subjected to an Any kD Mini-PROTEAN TGX Gel and transferred onto a PVDF membrane using the Transblot Turbo system (Bio-Rad). Blots were probed with a Can Get Signal and PVDF Blocking Reagent Set (TOYOBO). Every blot was also probed with an anti-actin antibody to ensure equal protein loading. Protein detection was achieved with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) using the Bio-Rad system (ChemiDoc XRS+).

We homogenized mouse tissues in buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P‐40, 0.5% deoxycholate, 0.1% SDS, and 1 mM 2‐mercaptoethanol with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, NJ, USA) and then centrifuged them at 2,500 × g for 15 min. Equal amounts of protein were separated by 5–20% SDS–PAGE and transferred to Hybond‐P membranes (GE Healthcare, Piscataway, NJ, USA). The primary antibodies and their dilutions were as follows: AR (N20, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Dnmt1 (1:1,000; Abcam, Cambridge, MA, USA), Dnmt3a (1:1,000; Abcam), Dnmt3b (1:1,000; Abcam), Hes5 (1:1,000; Santa Cruz), and ChAT (1:1,000; Millipore, Billerica, MA, USA). Primary antibody binding was probed with horseradish peroxidase‐conjugated secondary antibodies at a dilution of 1:5,000, and bands were detected by using an immunoreaction enhancing solution (Can Get Signal; Toyobo, Osaka, Japan) and enhanced chemiluminescence (ECL Prime; GE Healthcare). An LAS‐3000 imaging system (Fujifilm, Tokyo, Japan) was used to produce digital images. The signal intensities of these independent blots were quantified using IMAGE GAUGE software version 4.22 (Fuji) and expressed in arbitrary units (n = 3 for each group). The membranes were reprobed, or the same samples were examined with an anti‐GAPDH (1:5,000; Santa Cruz) antibody for normalization.