Nfatc1 monoclonal antibody

Western blotting was performed as previously described.³¹ (link) Monoclonal anti‐flag M2 antibody (F1804; Merck KGaA) was used to detect exogenous NFATC1. NFATC1 monoclonal antibody (MA3‐024, Thermo Fisher Scientific, Inc.) and NFATC2 monoclonal antibody (MA1‐025, Thermo Fisher Scientific, Inc.) were used to detect NFATC1 and NFATC2, respectively. DYRK1A polyclonal antibody (2771, Cell Signaling Technology, Inc.) was used to detect DYRK1A, and an anti‐β‐actin monoclonal antibody (A1978, Merck KGaA) was used to detect ACTB as a loading control. IRDye 680 goat anti‐rabbit IgG (c10207‐01, Li‐COR, Lincoln) and IRDye 800CW goat anti‐mouse IgG (C11026‐03, Li‐COR, Lincoln) were used as secondary antibodies. Detection and quantification were performed using the Li‐COR Odyssey imaging system.

U251 cells were seeded in a glass‐bottom dish. When cells were 30%–50% confluent, immunostaining was performed as previously described.³¹ (link) The cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% PBST. Then, the cells were blocked with 5% BSA and successively incubated with primary and secondary antibodies in 5% BSA–0.1% PBST. Finally, the stained cells were mounted in VECTASHIELD mounting medium with DAPI (H1200, VECTOR Labs), and the images were captured with LIONHEART FX (BioTek). NFATC1 monoclonal antibody (MA3‐024, Thermo Fisher Scientific, Inc.) was used to detect NFATC1. DYRK1A polyclonal antibody (2771, Cell Signaling Technology, Inc.) was used to detect DYRK1A. Mouse IgG (Santa Cruz, sc‐2025) and rabbit IgG (Proteintech, B900610) were used as negative controls. CoraLite488—conjugated Affinipure goat anti‐Mouse IgG (H+L) (SA00013‐1, Proteintech, Wuhan, Hubei, P.R.C) and CoraLite594—conjugated goat anti‐rabbit IgG (H+L) (SA00013‐4, Proteintech) were used as secondary antibodies.