Anti rabbit igg hrp linked ab

The GC cell line BGC-823 was purchased from the Shanghai Institute for Biological Sciences Chinese Academy of Sciences (Shanghai, China) and routinely maintained in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) in a 5% CO₂ humidified atmosphere at 37°C.
Rabbit anti-human DCTPP1 polyclonal antibody (pAb) (Cat#AP2821a) was obtained from Abgent (San Diego, USA). Mouse anti-human MDR1 (Cat# sc-55510) and mouse anti-DNMT1 monoclonal antibody (mAb) (Cat# sc-271729) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-α-tublin Ab (Cat# T6074) was purchased from Sigma (Louis, MO, USA). Rabbit anti-Phospho-BRCA1 Ab (Cat# 9009), rabbit anti-Phospho-H2A.X Ab (Cat# 2197), rabbit anti-caspase-3 (8G10) mAb (Cat# 9665), rabbit anti-cleaved caspase-3 (Asp 175) mAb (Cat# 9664), rabbit anti-Bax pAb (Cat#2772), rabbit anti-Bcl-2 (50E3) mAb (Cat# 2870), HRP-linked anti-rabbit IgG Ab (Cat# 7074) and HRP-linked anti-mouse IgG Ab (Cat# 7076) were purchased from Cell Signaling Technology (Beverly, MA, USA). dCTP, dTTP and 5-FU were purchased from Sigma and 5-methyl-dCTP was purchased from New England BioLabs (Ipswich, MA, USA).

Protein from MycCaP-Bo–derived bone metastasis with indicated treatments or cultured in vitro with various conditions was extracted by grinding tissues using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Roche). The concentration of protein was measured by BCA assay using Kit (Thermo Fisher Scientific). 50 μg of total protein was loaded into each well for SDS/PAGE and transferred to polyvinylidene difluoride membranes (Millipore). After blocking in Odyssey Blocking Buffer TBS (927-50000; LI-COR Biosciences), membranes were probed with primary Abs against pSRC (D49G4, #6943; Cell Signaling Technology) or SRC (36D10, #2109; Cell Signaling Technology) overnight at 4°C. Membrane was washed in Odyssey Blocking Buffer TBS three times and incubated with 680RD conjugated donkey anti-rabbit IgG Ab (926-68073; LI-COR Biosciences) for 1 h at room temperature or incubated with HRP-linked anti-rabbit IgG Ab (#7074; Cell Signaling Technology) for 1 h at room temperature. Membrane was detected using Infra-red Imager LI-COR Odyssey Fc chemi system.

Nuclear and cytoplasmic fractions were prepared from IEC lines using a Minute^TM cytoplasmic and nuclear extraction kit (Invent Biotechnology, Plymouth, MN, United States). Alternatively, whole cell lysates were prepared from human IEC lines or murine primary IECs as follows. Cells were washed with ice-cold PBS and incubated on ice for 30 min in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 60 mM n-octyl-β-D-glucoside, 1% Nonidet P-40) or RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS), both supplemented with protease inhibitor cocktail (Nacalai Tesque). After centrifugation at 4 °C and 20, 000 × g for 10 min, supernatants were collected. The obtained nuclear, cytoplasmic, or whole cell lysates were subjected to western blotting using a combination of anti-RBM14 Ab (GTX112293, GeneTex, Irvine, CA, United States; ab70636, Abcam) as the primary antibody and HRP-linked anti-rabbit IgG Ab (Cell Signaling Technology) as the secondary Ab, or HRP-linked anti-β-actin Ab (ab49900, Abcam). After chemiluminescent detection, band intensities were calculated using ImageJ software.