Quantifying RNA Transcript Abundance

Culture media was removed, cells were washed with ice-cold ACSF, and RNA was isolated by phenol-chloroform extraction (TRIzol, Invitrogen). Relative transcript abundance was quantified by two-step RT-PCR (Quantitect SYBR Green, Qiagen) using the primers listed in Table 5. Primers for NFATc3 and NFATc4 (Vashishta et al., 2009 (link)), and for GAPDH (Garcia et al., 2021 (link)) were previously published and validated. Primers for Mdm2 were designed using NCBI Primer-BLAST and validated prior to use. Fold-change was calculated using the 2^−ΔΔCt method, commonly referred to as the “delta-delta Ct method”.