Anti mouse igg peroxidase

The aptamers used in all the experiments were synthesized by IDT (Coralville Johnson County, IA, USA). Recombinant human glypican proteins (GPC3 and GPC5) were purchased from R&D Systems (Minneapolis, MN, USA), and anti-human GPC3 monoclonal human antibody was purchased from G&P Bioscience (Santa Clara, CA, USA). HSA, BSA, human serum, and anti-mouse IgG peroxidase were purchased from Sigma (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated streptavidin was purchased from Thermo Fisher Scientific (Minneapolis, MN, USA). The following buffers were used: TBS-T buffer (0.1% (v/v) Tween 20 in Tris-buffered saline, pH 7.2), blocking buffer (5% non-fat dried milk in TBS-T), and washing buffer (TBS-T buffer).

Total proteins were extracted in RIPA buffer containing 1 mM sodium orthovanadate and Complete Protease Inhibitor Cocktail (Roche Applied Science) and quantified using the Pierce™ BCA protein assay kit (Thermo Fisher). Equal amounts of proteins were loaded onto SDS–polyacrylamide gels and transferred to PVDF membrane. The membranes were then blocked with 5% non‐fat milk in TBST 0.1% and immunoblotted overnight at 4°C with the following primary antibodies (1:1,000): TCHP (Santa Cruz Biotechnology, SC‐515025), β‐actin (Sigma, A5441), p62 (GeneTex, GTX100685), LC3B (CST, #2775), p16 (BD Biosciences, 551154), NF‐κB (Millipore, MAB3026), (S536) NF‐κB (CST, #3033), PCM1 (CST, #5213) and GABARAP (CST, #13733), NDP52 (CST, #60732) and V5 (Thermo Fisher, R960‐25). Secondary antibodies (1:5,000): anti‐Mouse IgG–Peroxidase (Sigma, A5906) and anti‐Rabbit IgG–Peroxidase (Sigma, A0545), were incubated for 1 h at RT. Pixel intensity/quantification was performed using ImageJ.

Total proteins were prepared by resuspending 1 × 10⁶ cells in extraction buffer (50 mM Tris-HCl pH 7.6; 0.15 M NaCl; 5 mM EDTA; 16 Protease Inhibitors; 1% Triton X-100). One 40 s pulse of sonication (UP100H manual sonicator, Hielscher) at 40% amplitude was performed to allow dissociation of protein from chromatin and solubilization. Extracts were analyzed by SDS-PAGE using an 8% gel (37.5:1 Acryl/Bis Acrylamide). The following primary antibodies were used: Beta-Actin (Santa-Cruz sc1616, rabbit 1:4000), H3 total (Abcam ab1791, rabbit 1:6000), Lamin A/C (Santa Cruz sc-6215, goat 1:4000), Lamin B (Santa Cruz sc6216, goat 1:2000), progerin (13A4 mouse, Abcam 66587, mouse 1:1000), Ezh2 (AC22 Cell Signaling 3147S, mouse 1:1000), Bmi1 (D42B3 Cell signaling, rabbit 1:1000), H3K9me3 (Abcam ab8898, rabbit 1:1000), H3K27me3 (Millipore 07-449 rabbit 1:1000). HRP-conjugated secondary antibodies were revealed with the ECL chemiluminescence kit (ThermoFisher Scientific). The following secondary antibodies were used: Anti-Mouse IgG-Peroxidase (Sigma, A9044), Anti-Rabbit IgG-Peroxidase (Sigma, A9169), Anti-Goat IgG-Peroxidase (Sigma, A5420).

Recombinant PDC-E2, BCOADC-E2, and OGDC-E2 were coated overnight at 4 °C at a concentration of 15 μg/ml in a 96-well-plate. The plates were subsequently blocked with 3% skimmed milk for 1 h. Serum diluted 1/20 was added to the plates for 1 h. Anti-mouse IgG peroxidase (Sigma-Aldrich) was subsequently added to the plates for 1 h. Finally, a TMB Substract Reagent Set (BD Biosciences) was added for 10 min. Following the addition of stop solutions (BD Biosciences), the optical density was read at 450 nm.

The EA lysates (20 μg) were incubated with 4 × sample buffer and subjected to SDS–PAGE (Laemmli, 1970 (link)). Proteins from the gel were transferred to nitrocellulose membranes to perform the immunoblotting (Towbin et al., 1979 (link)). Membranes were blocked for 1 h with ECL blocking agent (GE Healthcare), TBS (50 mM Tris–HCl, 150 mM NaCl, pH 7.4), and TBS-T (TBS with 0.1% Tween 20). Then, nitrocellulose membranes were incubated with mAb1D9 (1:500, ascites), mAb2C2 (1:500 ascites), MAP2 (1:500, mouse antiserum), or anti-α-tubulin (1:1000, Sigma-Aldrich) overnight with 5% bovine serum albumin (Sigma-Aldrich) in TBS-T. Next, the membranes were incubated for 1 h with a secondary antibody, anti-mouse IgG-peroxidase (Sigma-Aldrich), and visualized by a chemiluminescence detector (UVITEC) in the presence of luminol with the ECL Prime Western blotting detection reagent (GE Healthcare).