Anti gfp antibody

Cells were harvested 24 h after transfection or infection with expression vectors and subjected to SDS-PAGE and Western blotting under the conditions described previously [11 (link)]. A rabbit anti-human REIC/Dkk-3 antibody was raised in our laboratory [11 (link)]. Goat anti-human KLF16 antibody (Abcam, Inc., Cambridge, MA), anti-6x histidine antibody (MBL Co., Nagoya, Japan), anti-HA antibody (Cell Signaling Technology, Danvers, MA), and anti-GFP antibody (Clontech) were purchased.

Transiently transfected COS-7 cells were washed in PBS and fixed with 4% paraformaldehyde. Surface expression of NMDARs was achieved by immunolabeling extracellular GFP (GFP cloned in-frame within GluN2 subunits ATD), incubating with anti-GFP antibody (Clontech) for 1 h at RT under non-permeabilizing conditions. After washing, cells were incubated with anti-rabbit IgG-Alexa555 secondary antibodies (Life Technologies, Carlsbad, CA, USA), for 1 h at RT. The total amount of GFP-tagged GluN subunits was detected by the GFP endogenous fluorescent signal emitted by GFP-GluN2A/GluN2B constructs. Coverslips were mounted in ProLong antifade mounting medium (Life Technologies) and images were acquired in a Nikon Eclipse 80i microscope (63×/1.4 N.A. immersion oil objective).

GFP-ubiquitin tagged cells were cross-linked with 1 % formaldehyde for 10 min and lysed in buffer IP1 (10 mM Hepes-KOH pH 7.5, 10 mM NaCl, 3 mM CaCl₂, 0.25 M sucrose, 1 mM DTT, 1 mM PMSF). After 30 min on ice, nuclei were collected by centrifugation at 3000 g for 10 min. The pellet was lysed in IP2 buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS) and sonication 10 times at 40 % 10 sec (Branson). Immunoprecipitation was carried out with anti-GFP antibody (Clontech, Mountain View, CA, USA) or irrelevant immunoglobulin G (Sigma, St. Louis, MO, USA) in IP buffer (16.7 mM Tris pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1 % Triton X-100, 0.01 % SDS). Samples were then processed as indicated in EZ ChIP™ Kit (Upstate, Cell Signaling Technology, Danvers, MA, USA). DNA was purified using GFX PCR DNA purification kit (Amersham, GE Healthcare Europe GmbH, Freiburg Germany). Promoter regions were detected by qPCR with SYBR Green PCR master mix (Applied Biosystems, Life Technologies, Paisley, UK). ChIP results were quantified relative to the input amount. Chromatin immunoprecipitation primers for human ΔNp63 promoter were 5'-GGTTGGCAAAATCCTGGA-3'; 5'-TCACTAAATTGAGTCTGGGCATT-3'.

Protein samples were denatured by boiling at 95°C for 3 min in protein-loading buffer [2% (w/v) SDS, 100 mM DTT, 0.05% (v/v) BPB, and 10% (v/v) glycerol], resolved by SDS-PAGE, and transferred to 0.2-μm polyvinylidene difluoride membrane (Wako) using the semi-dry system (Trans-blot Turbo, Bio-Rad). The membrane was blocked in 4% (w/v) skim milk (Nacalai) in 1× phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween 20 and further incubated with the following antibodies: anti-Aub antibody (1:500; guinea pig) (80 (link)), anti-GFP antibody (1:2000; Clontech, rabbit), anti-Ago1 antibody (1:1000; Abcam, Ab5070, rabbit), anti-Ago2 antibody (1:100; guinea pig) (25 (link)), anti-Piwi (1:10; mouse, P4D2), anti-FLAG M2-peroxidase [horseradish peroxidase (HRP)] (1:5000; Sigma-Aldrich, #A8592), anti-H3K18Ac (1:2000; Active Motif, #39756), anti-H3K27Ac (1:2000; Active Motif, #39136), anti-H4K8Ac (1:2000; Active Motif, #61104), anti-H4K12Ac (1:2000; Active Motif, #39928), anti-guinea pig immunoglobulins-HRP (1:1000; Dako), anti-rabbit immunoglobulin G (IgG)–HRP (1:3000; Bio-Rad), anti-mouse IgG-HRP (1:3000; Bio-Rad). The chemiluminescent signals were obtained by using Chemi-Lumi One (Nacalai) and detected by Chemidoc MP Imaging system (Bio-Rad). The images were processed by using Pixelmator or Fiji. The immunoblot signals were quantified by using Fiji.

Ten-day-old seedlings were ground in liquid nitrogen and resuspended in 1 ml of extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% NP-40, 10% glycerol and protease inhibitors). The extract was cleared by successive centrifugation for 5 min at 5,000 g, 5 min at 10,000 g and 1 hr at 16,000 g. The soluble fraction was subsequently concentrated at 14,000 rpm in YM-10 centricons (Amicon). About 250 μL (1 mg) of cleared extract was injected in a pre-calibrated Superdex 200 gel filtration column (GE Healthcare) and run with the same extraction buffer at 0.4 ml.min-1 in an AKTA FPLC system. Twenty 0.5 ml fractions were collected and analyzed by immunoblotting with an anti-GFP antibody (632381 Clontech).