Hrp conjugated anti mouse igg

Anti-dsDNA antibodies were measured as following: 96-well Nunc MaxiSorp plates (Thermo Scientific) were irradiated under UV light for 1 h and then coated with calf thymus DNA (5 μg/ml, Invitrogen) in DPBS. After overnight incubation at 4°C, plates were blocked in 1% BSA in DPBS and incubated in a wet chamber for 1 h at room temperature. Plasma samples were added at 1:80 (IgM) or 1:200 (IgG) dilutions and incubated for 2 h at 37°C. For detection, biotinylated rat anti-mouse IgM (1:2000, BD Pharmingen) or HRP-conjugated anti-mouse IgG (1:1000, GE Healthcare) was added and incubated for 1 h at 37°C. For anti-dsDNA IgM measurements, streptavidin-HRP (R&D Systems) was added in a 1:200 dilution for 30 min at 37°C. Samples were developed using TMB substrate solution according to the manufacturer's instructions. The reaction was stopped with 1M H₂SO₄ (Honeywell) and the absorbance was measured with a plate reader at 450 nm as the primary wavelength.
Total IgM, IgG1, IgG2b, IgG2c, IgG3, and IgA levels were measured by a chemiluminescence-based sandwich ELISA as described previously (19 (link)). IgE levels were determined using a mouse IgE-specific ELISA (BioLegend). MDA-LDL was prepared as described previously (19 (link)). Antigen-specific antibody titers were measured by chemiluminescent ELISA as previously described (20 (link)).

Each fractions were added to 4× NuPAGE sample buffer (Life technologies Carlsbad, CA, USA), heated at 70°C for 10 min, and electrophoresed in 12.5% polyacrylamide gels (Bio Craft, Tokyo, Japan), and then electroblotted to 0.2 μm PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked with 2.5% skimmed milk (Nacalai tesque) in 1× TBS-T (Sigma Aldrich) for 1 h at room temperature. After blocking, the blots were incubated with a monoclonal anti-tau antibody HT7 (1:1,000, Thermo Fisher Scientific, Waltham, MA, USA) in blocking solution for 2 h at room temperature. The blots were washed with 1× TBS-T for 30 minutes. The washed blots were incubated with HRP-conjugated anti-mouse IgG (1:2000, GE healthcare) for 1 h at room temperature, and washed as described above. Tau proteins were detected by chemiluminescent HRP substrate (Merck Millipore) and analyzed by using an imaging analyzer ChemDoc (Bio-Rad). For preparation of standard curve to calculate the amount of tau, the 2-fold serial dilutions of sarkosyl-insoluble fraction were included in each gel. The standard dilutions were prepared by mixing some of the WB samples, which were preliminarily selected by WB of some severe-clasping phenotype samples. The samples outside the range of the standard curve were properly diluted and re-assayed.

HeLa S3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (Sigma). Anti-RRP4 (ab156698) antibody was purchased from Abcam. Anti-α-tubulin (B-5-1-2) antibody was purchased from Sigma. Anti–Pol II (N20) antibody was purchased from Santa Cruz Biotechnologies. HRP-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from GE Healthcare. SSA was prepared as described previously [4] (link). Control siRNA (siGENOME Non-Targeting siRNA Pool #1) and RRP4 siRNA were purchased from Thermo Scientific. siRNA transfection was performed using Lipofectamine RNAiMAX Reagent (Life Technologies). The sequences of U1, U2, and control AMOs were 5′-GGTATCTCCCCTGCCAGGTAAGTAT-3′, 5′-TGATAAGAACAGATACTACACTTGA-3′ and 5′-CCTCTTACCTCAGTTACAATTTATA-3′, respectively; AMOs were purchased from Gene Tools, LLC. AMO transfection was performed using the Neon Transfection System (Life Technologies). HeLa cells (5×10⁵ cells) were trypsinized, washed twice with PBS, and resuspended in 100 µl of the resuspension buffer. After mixing cells with AMOs, electroporation was performed with the following parameters: 1300 V, 10 msec, 3 pulses.

Mouse monoclonal anti-α-tubulin (T6199) was purchased from Sigma-Aldrich. Rabbit polyclonal anti-p27 (N-20) (#sc-527) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). HRP-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from GE Healthcare (Little Chalfont, UK).
For immunoblotting, cells were suspended in lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl₂, 1 mM EGTA, 1 mM EGTA, 1% Nonidet P-40, 10% glycerol, cOmplete ULTRA tablets mini EDTA-free [Roche, Basel, Switzerland] and PhosSTOP [Roche]) and vortexed for 15 s. Cell lysates were subjected to immunoblotting with the indicated antibodies. Immune complexes were detected using the NOVEX ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) on an ImageQuant LAS 4000mini (GE Healthcare).

FM3A, MTT060562, and 3T3 cells, before and after treatment with transduction proteins, were used for western blotting. The samples were loaded onto a 5%–20% gradient polyacrylamide gel (Wako, Tokyo, Japan) and electrophoretically transferred to nitrocellulose membranes (GE Healthcare, Danbury, CT, USA). The membranes were blocked with 10% skim milk in PBS. The primary antibodies were anti-FLAG M2 mouse monoclonal antibody (Sigma Aldrich), anti-HA mouse monoclonal antibody (Sigma Aldrich), anti-MCM2 mouse monoclonal antibody (BD Biosciences), anti-DNA-PK_cs mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-DNA-PK S2056 (Mouse-S2053) rabbit polyclonal antibody (Assay Biotech, Sunnyvale, CA, USA), anti-P53 mouse monoclonal antibody (Merck, Darmstadt, Germany), anti-phospho-P53 (Ser 15) rabbit polyclonal antibody (Merck), anti-cleaved caspase-3 rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-GFP mouse monoclonal antibody (Abcam), anti-PP2A rabbit polyclonal antibody (Cell Signaling Technology) and anti-GAPDH rabbit polyclonal antibody (Santa Cruz Biotechnology). The secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GE Healthcare) and HRP-conjugated anti-rabbit IgG (GE Healthcare). Protein expression was detected using the Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA).

The adult flies were homogenized in SDS sample buffer (12.5 mM Tris (pH 6.8), 20% glycerol, 4% SDS, 2% 2-mercaptoethanol, and 0.001% bromophenol blue) and boiled for 10 min at 95°C. The samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Buckinghamshire, UK). After blocking with 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), the membranes were incubated with a primary antibody in Tris-buffered saline (TBS) containing Tween-20 (TBST) overnight at 4°C and then with a secondary antibody in TBST for 1 h at 25°C. The signals were detected with an ECL-plus kit (GE Healthcare). As primary antibodies, rabbit anti-phospho-Akt antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-p70 S6 kinase (Cell Signaling Technology), and mouse anti-α-tubulin (Sigma-Aldrich) were used at dilutions of 1:1000, 1:1000, and 1:5000, respectively. HRP-conjugated anti-rabbit IgG (Cell Signaling Technology) and HRP-conjugated anti-mouse IgG (GE Healthcare) were used as secondary antibodies at dilutions of 1:2000 and 1:1000, respectively.

Transfected HEK293T cells were washed with PBS, and lysed directly with RIPA buffer containing a protease inhibitor cocktail (Roche). After sonication and centrifugation, the supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-P (GE Healthcare). The blots were probed with an appropriate primary antibody, followed by HRP-conjugated anti-mouse IgG (GE Healthcare). The protein signals were detected using ECL prime Western blot detection reagent (GE Healthcare). The images were analyzed using an ImageQuant Las 4000 mini (GE Healthcare).