Enhanced chemiluminescence ecl western blotting detection reagents

Total protein levels in HPDL cells were determined by immunoblotting. The HPDL cells were washed by PBS followed, then after, lysed using RIPA (Radio-Immunoprecipitation Assay) buffer for 30 min. And lysates were centrifuged at 12,000 rpm for 15 min. Afterwards, Bradford (Sigma Alrich, St. Louis, MO, USA) analysis was performed to quantify protein was performed. Then, the equal amount of protein (20 μg) was separated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% (w/v) skim milk dissolved in TBS-T buffer (10 mM Tris (pH 8.0) and 150 mM NaCl) at room temperature for 1 h. After blocking, the membranes were rinsed with TBST buffer for 1 h and incubated with primary antibody overnight at 4 °C. The membranes incubated the primary antibody were washed with Tris Buffered Saline with Tween 20 (TBS-T) buffer and then incubated in a secondary antibody. The protein bands were detected with Enhanced Chemiluminescence (ECL) Western blotting detection reagents (Thermo Fisher Scientific; Waltham, MA, USA). The band images were scanned with an ChemiDoc XRS+ system (Bio-Rad, CA, USA). The detected bands were quantified using the image J 1.54i version software (National Institutes of Health, Bethesda, MD, USA).

MTT powder, PMA, and A23187 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Annexin V and caspase 3/7 staining reagents for the IncuCyte^® cell imaging system were obtained from Essen Bio (Ann Arbor, MI, USA). Human IL-2 ELISA kits were purchased from R&D (Minneapolis, MN, USA). CFSE dye, enhanced chemiluminescence (ECL) Western blotting detection reagents, and the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The polyvinylidene fluoride (PVDF) membrane was purchased from Bio-Rad (Hercules, CA, USA). Anti-β-actin, anti-cyclin A, and anti-cyclin E antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-CD3 and anti-CD28 antibodies were purchased from Bioxcell (West Lebanon, NH, USA). Anti-CD69 and anti-CD25 conjugated with APC were obtained from eBiosciences (San Diego, CA, USA). Anti-caspase 3, anti-caspase 7, anti-caspase 8, anti-caspase 9, anti-TAK1, anti-IKKα, anti-p65, anti-poly (ADP-ribose) polymerase (PARP), anti-IκBα, anti-ERK, anti-p38, anti-JNK, and anti-c-Jun were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phosphorylated TAK1, IKKα, ERK, p38, JNK, and c-Jun antibodies were also obtained from Cell Signaling Technology.