Ab54073

Neonatal cardiomyocytes in 4-well chambers were washed with phosphate-buffered saline (PBS) three times, fixed with 4% paraformaldehyde for 15 minutes, permeabilized in 0.3% Triton X-100 for 10 minutes, and blocked with 3% bovine serum albumin for 1 hour at room temperature. The following primary antibodies were used: Lats2 (1:200 dilution, #ab54073) (Abcam), FoxO1 (1:100 dilution, #1874-1) (epitomics), YAP (1:100 dilution, H00010413-M01) (Abnova), and α-actinin (1:400 dilution, A7811) (Sigma). Alexa Fluor 488 Dye- or Alexa Fluor 594 Dye-conjugated secondary antibody (Invitrogen) was used for detecting indirect fluorescence. Slides were mounted using a reagent containing DAPI (VECTASHIELD; Vector Laboratories Inc.).

Antibodies used for immunoblots were purchased from the indicated companies: Catalase (1:3000 dilution, #ab1877) (Abcam), p-Akt (Ser473) (1:2000 dilution, #9272), Akt (1:2000 dilution, #9271), p-eNOS (Ser1177) (1:1000 dilution, #9571), eNOS (1:1000 dilution, #9586), p-FoxO1 (Thr24, Ser256) (1:1000 dilution, #9464 and #9461), FoxO3 (1:1000 dilution, #9467), GAPDH (1:3000 dilution, #2118), Histone H3 (1:500 dilution, #9715), Lamin A/C (1:5000 dilution, #4777), p-Mst1 (Thr183) (1:1000 dilution, #3681), p-YAP (Ser127) (1:1000 dilution, #4911), YAP (1:2000 dilution, #4912) (Cell Signaling Technology), FoxO1 (1:2000 dilution, #1874-1) (epitomics), α-tubulin (1:5000 dilution, #T-6199), FLAG (1:2000 dilution, #F3165) (Sigma), MnSOD (1:3000 dilution, #611580), Mst1 (1:2000 dilution, #611052) (BD Biosciences), and Lats2 (1:1000 dilution, #ab54073 and #A300-479A) (Abcam and Bethyl Laboratories). The p-Lats2 (S872 and T1041) (1:500 dilution) antibodies were generated as described^{41 (link)}.