Chemiluminescence reagents

Proteins were prepared by using the Cell Lysis buffer (Tris-HCl) (Bioss, Beijing, China). The concentrations of proteins were measured with the Bicinchoninic Acid (BCA) assay method (Meilunbio, Dalian, China). Ten micrograms of proteins were separated by electrophoresis through SDS-polyacrylamide gels (YEASEN, Shanghai, China). The proteins obtained were then transferred to a polyvinylidene fluoride (PVDF) (Merck, Shanghai, China) membrane. The membrane was blocked with a rapid blocking solution for 2 h and then incubated with primary antibodies at 4 °C for 14 h. Subsequently, the PVDF membrane was washed and then hatched with a second antibody for 2 h. Finally, the protein bands were visualized with chemiluminescence reagents (Meilunbio, Dalian, China) and quantified using the ImageJ2 program. The CyclinD1 (CCND1) and ADAR1 antibodies were purchased from Aifang biological; Proliferating Cell Nuclear Antigen (PCNA), Bcl-2-associated X protein (Bax) and cysteine-dependent aspartate specific protease 3 (Caspase-3) antibodies were from Prteintech; B-cell leukemia/lymphoma-2 (Bcl2) was from Zenbio (Chengdu, China); β-actin antibody was from Cell Signaling Technology (Danvers, MA, USA).

We used western blots to investigate the effects of palbociclib on the activation of the FcεRI signaling regulatory proteins Lyn and the MAPK signaling proteins p38, JNK, and ERK1/2, which have bene reported to be involved in mast cell activation [26 (link)]. BLCs were sensitized with 50 ng/ml DNP-IgE overnight and washed twice with PBS and placed in fresh media. After incubating with or without palbociclib at 37 °C for 1 h, BLCs were stimulated with 100 ng/ml DNP-HSA and then washed twice with PBS. Cells were lysed in RIPA buffer (Beyotime, Beijing, China) with a protease-inhibitor cocktail (Med Chem Express, Monmouth Junction, NJ). Cell lysates were centrifuged at 12,000 rpm for 15 min. Supernatants were mixed with a loading sample buffer (Thermo Fisher Scientific) and denatured by heating for 10 min at 100 °C. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore, Billerica MA). After incubating with a primary antibody in tris-buffered saline with 0.1% tween 20 buffer that contained 5% bovine serum albumin or skim milk. The membranes were incubated with horse-radish peroxidase-conjugated secondary antibodies for 1 h at room temperature in the same buffer. Chemiluminescence reagents (Meilun, Dalian, China) were applied according to the manufacturer’s protocol.