Hrp conjugated igg secondary antibody

The cultured cells were washed with PBS 2–3 times, and 150 μL of RIPA Buffer was added. The cells were lysed for 30 min and centrifuged at 12,000×g for 10 min at 4 °C. Protein concentration was quantified using standardizing BSA (bovine serum albumin). Ten μg of lysate was denatured with 10% Mini-protean TGX™ and transferred to a polyvinylidene difluoride (PVDF) membrane at 100 V for 1 h. The membrane was blocked with TBST (0.1% Tween 20 + TBS) solution containing 5% skim milk for 1 h. The primary antibody was diluted with skimmed milk (1:1000) and the reaction was allowed to occur overnight at 4 °C, after which washing was carried out 3 times using TBST. The HRP secondary antibody (horseradish peroxide (HRP) conjugated IgG secondary antibody (Cell Signaling, #5157, 1:2000) was diluted 1: 1000 times, allowed to react for 2 h at 4 °C, washed three times with TBST, and allowed to react with the ECL substrate. Protein levels were detected with a specific antibody, using the ChemiDoc™ imaging systems (Bio-Rad, Hercules, CA).

For 5hmC detection, genomic DNA samples were denatured and twofold serial dilutions were spotted on a nitrocellulose membrane (#88014, Thermo Fisher). The blotted membrane was UV-cross-linked (70,000 μJ/cm²) and blocked with 5% skimmed milk powder for 1 h at room temperature and incubated with anti-5hmC antibody (dilution 1:1000; #39769, Active Motif) at 4 °C overnight. The membrane was subjected to immunoblot analysis using HRP-conjugated IgG secondary antibody (dilution: 1:10000; #7074, Cell Signaling Technology) for 1 h at room temperature. To ensure equal spotting of total DNA on the membrane, the blot was stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2).

For western blot analysis, frozen tissue samples were pulverized under liquid nitrogen, and tissue homogenization was performed with TissueLyser II (QIAGEN). Tumors were lysed in a RIPA buffer. After protein isolation, protein samples (10-40 μg) were separated on 10% SDS polyacrylamide gels and electrotransferred onto nitrocellulose membranes. After blocking, the membranes were incubated with primary anti-human and anti-mouse CD13 (from Santa-Cruz Biotechnology, Inc., USA) antibody at the dilution of 1 : 1000 overnight at 4°C. After washing, the membranes were probed with IgG HRP conjugated secondary antibody (Cell Signaling Technology, Inc., Beverly, MA, 1 : 2000). Beta-actin was used as a loading control. Bands were visualized by enhanced chemiluminescence reaction. Densitometry was performed using the ImageJ software. For the detailed protocol, please see Supplementary Material western blot analysis.

A20 lymphoma tissue was minced with a bead beater (TissueLyser II, Qiagen). Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% TritonX 100, 0.5% sodium deoxycolate, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, protease inhibitor cocktail). Protein isolation, SDS-PAGE, and Western blotting were performed according to Nagy et al. [41 (link)]. Protein samples (10–40 µg) were separated on 10% SDS polyacrylamide gels and electrotransferred onto nitrocellulose membranes. After blocking for 1 h with TBST containing 5% BSA, the membranes were incubated with the primary antibodies against CAIX and CAXII overnight at 4 °C (dilution: 1:1000) or with TBST. After washing with 1 x TBST solution, the membranes were probed with IgG HRP conjugated secondary antibody (Cell Signaling Technology, Inc. Beverly, MA, 1:2000). Bands were visualized by enhanced chemiluminescence reaction (SuperSignal West Pico Solutions, Thermo Fisher Scientific Inc., Rockford, USA).

Western blotting was performed as described previously [22 (link)]. Tissues were homogenized in ice-cold PRO-PREP protein extraction buffer (iNtRON Biotechnology, Seongnam, Republic of Korea), and centrifuged at 16,600 g for 10 min at 4 °C. Supernatants were collected, boiled, and analyzed by an SDS-PAGE-immunoblotting assay. Antibodies against GAPDH, phospho-IKKα/β (Ser176+Ser180), IKKα (Bioss Antibodies, Woburn, MA, USA), NF-κB p100/p52, NIK, RelB (Cell Signaling Technology, Berverly, MA, USA), phospho-STAT3 (Tyr705) (Cambridge Bioscience, Cambridge, UK) and UCP1 (Abcam, Cambridge, UK) were used as primary antibodies, followed by the appropriate IgG-HRP conjugated secondary antibody (Cell Signaling Technology). Proteins were visualized by ECL.