Ab99431

Immunofluorescence on human carotid artery sections and mice aortic sinus sections was performed. Briefly, for double-staining, sections were incubated with anti-α-SMA antibody (ab240654, Abcam), together with anti-NFATc1 antibody (ab25916, Abcam), anti-NFATc2 antibody (sc-7296, Santa Cruz), anti-NFATc3 antibody (sc-8405, Santa Cruz) or anti-NFATc4 antibody (ab99431, Abcam) at 4°C overnight, followed by a 30-min incubation with secondary antibody conjugated to Alexa Fluor 594 (R37121, Molecular probes) and Alexa Fluor 488 (R37116, Molecular probes). The signals of individual and merged images for antigen detection were performed using a fluorescence microscope (Olympus, Japan) and Axiovision 4.8 software.

Western blotting was performed as previously described (20 (link)). Equal amounts (20 µl) of sample buffer were loaded onto a 10% SDS-PAGE gel. The protein was electrotransferred to polyvinylidene difluoride (PVDF) membranes. Then, the membranes were incubated with the following primary antibodies at 4°C for 12 h: Anti-inhibitors against NFATc4 (ab99431, 1:1,000; Abcam), VCAM-1 (ab134047, 1:1,000; Abcam), ICAM-1 (10020-1-AP, 1:1,000; Proteintech), IL-8 (ab7747, 1:1,000; Abcam), TNF-α (17590-1-AP, 1:1,000; Proteintech), Lamin B (AF1408, 1:1,000; Beyotime Institute of Biotechnology) and β-actin (20536-1-AP 1:1,000; Proteintech). The membranes were incubated at room temperature for 1 h with a peroxidase-conjugated secondary antibody (1:2,000; Beyotime Institute of Biotechnology). The protein bands were visualized using a ChemiDoc imaging system (Bio-Rad Laboratories, Inc.), and the intensity was quantified using ImageJ 1.8.0 (National Institutes of Health).