Hrp conjugated goat anti rabbit igg antibody

WB analysis was performed as previously described [29 (link)] to confirm the expression of CtBP2 and GAPDH in the GC cell lines, GC tissues, and matched adjacent normal tissues. Tissue samples for WB were stored in N₂. RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) was used to extract total protein from tissue samples and cell lines. Protein concentration was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein were separated using 10% SDS/polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime Institute of Biotechnology) and were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were probed with an anti-CtBP2 antibody (1:20,000; Abcam, Cambridge, UK). Expression of CtBP2 was determined using a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:1,000; Proteintech, Rosemont, IL, USA). After stripping, the membranes were reprobed with an anti-GAPDH mouse monoclonal antibody (1:1,000; Proteintech) overnight at 4°C as a loading control. The bands were visualised using an ECL system (Thermo Fisher Scientific Inc., Waltham, MA, USA) and quantified by densitometry.

HEK293 cells were transfected with pBlueScript vector, or the individual HBoV2 genomic recombinant plasmids. At 72 h post-transfection, the cells were lysed on ice for 30 min in lysis buffer (25 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1% NP-40 or 1% Triton X-100, pH 7.4) supplemented with a protease inhibitor cocktail (Roche, 04693132001). The cell lysates were centrifuged at 12,000 rpm at 4°C for 15 min to remove insoluble material. Aliquots (100 μg) of the total protein contents separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis were transferred to nitrocellulose membranes (Pall, 66485). After blocking with 5% nonfat dry milk solution in PBS (RT, 1 h), the membranes were incubated with rabbit anti-HBoV2 NS1 (1:1000) (Jiaxuan Biotech, China) at 4°C overnight. The following day, the membranes were washed three times in PBST and incubated with HRP-conjugated goat anti-rabbit IgG antibody (Proteintech, 1:4000) at RT for 2 h. The bands were visualized by ECL with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA).

Cells were pre-stimulated with pimecrolimus (30 μmol/L), a CaN inhibitor, or rMgPa (30 μg/mL) for 30 min and then incubated with Phorbol 12-Myristate 13-Acetate (PMA)/ionomycin (500 ng/mL PMA and 10 μg/mL Ionomycin). Simultaneously, cells were transfected with CypA-siRNA (Origene Technologies) using Lipofectamine 3,000 according to the manufacturer's instructions. Cells were then harvested and solubilized for 1.5 h at 4°C in lysis buffer containing a protease inhibitor mixture. CaN was isolated using SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. The primary and secondary antibodies were designated as the phosphorylated CaN antibody (1:200, Abcam) and the HRP-conjugated goat anti-rabbit IgG antibody (Proteintech), respectively. CaN phosphorylation was detected by WB using a chemiluminescent substrate using ImageJ software.