Antibody for β actin a1978

HaCaT cells were plated at a density of 5 × 10⁵ cells/well in a 6-well plate and cultured in a 5% CO₂ incubator at 37 °C for 24 h. Then, DPM (25 μg/mL) and PAE or FQA were treated with cells and incubated for another 24 h. The same amount of extraction vehicle (butylene glycol for PAE, or solvent DMSO for FQA) was added to the control. The cells were washed twice with cold PBS and lysed with RIPA buffer containing protease inhibitor cocktail. The lysates were centrifuged at 4 °C, 13,000 rpm for 15 min. Supernatants were harvested, and protein quantification was performed by Bradford analysis. Samples with equal amounts of protein (20 μg) were separated using 4–12% SDS polyacrylamide gels, transferred to polyvinylidene difluoride membranes (PVDF), and blocked with 5% BSA in TBST buffer for 25 °C for 1 h. Antibody dilutions were as follows: PAR-2, 1:1000; occludin, 1:1000; ZO-1, 1:1000; β-actin, 1:10,000. Antibodies specific for PAR-2 (#6976), occludin (#91131), and ZO-1 (#13663) were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody for β-actin (A1978) was from Sigma Aldrich (St. Louis, MO, USA). Detection of bands was performed using Image Quant LAS 4000 (GE Healthcare, IL, USA) hardware and software, according to the manufacturer’s instructions. Signal intensities were quantified by densitometry, with Image J (NIH, Bethesda, MD, USA).