Ab14613

To investigate PDE4D protein localization, HT22 cells were seeded on 12 mm glass coverslips (VWR, 631–1577) coated with 100 μg/mL Poly-l-Ornithine (Sigma, P4957) and 1 μg/mL laminin (Sigma, L2020) and grown for 24 h. After fixation in 4% paraformaldehyde, cells were permeabilized using 0.1% Triton X-100. After blocking with 10% BSA for 1 h, cells were incubated with rabbit anti-PDE4D (1:250; ab14613, Abcam) overnight at 4 °C. Then, cells were incubated with goat anti-rabbit Alexa647 (1:250; Invitrogen) for 1 h at room temperature. Finally, nuclei were counterstained with Hoechst (1:500; Sigma).
For immunocytochemistry following transfection experiments in HT22, the same protocol was used except for the antibodies used. Mouse anti-FLAG primary antibodies (1:1000; M2 clone, Sigma-Aldrich) and donkey anti-mouse Alexa488-conjugated secondary antibody (1:250; Invitrogen) were used to determine which cells were successfully transfected and expressed the FLAG-encoding PX458 plasmid. PDE4D localization was imaged after mounting the coverslips on microscope glasses using a disk spinning unit (DSU) microscope (Olympus). Morphology assessment of transfected, FLAG-positive HT22 cells was performed as described above.

Male and female mouse (C57BL/6J) ventricular tissue samples from the RV base and apex were lysed in radioimmunoprecipitation assay buffer with phosphatase and protease inhibitors. Total proteins (50 μg per sample) were separated on SDS–polyacrylamide gel electrophoresis gel and then transferred onto a polyvinylidene difluoride membrane. Membrane was incubated with primary antibody overnight at 4°C and then labeled with IRDye 800CW– or 680RD-conjugated secondary antibodies at room temperature for 1 hour. Images were scanned with ChemiDoc MP Imaging System (Bio-Rad Laboratories, CA), quantified by Image Lab software, and normalized to γ-tubulin. The following primary antibodies were used for immunoblotting: PDE3A (112AP, FabGennix International Inc., Frisco, TX), PDE4A and PDE4B (gifts from M. Conti, University of California at San Francisco), PDE4D (ab14613, Abcam, Cambridge, MA), and γ-tubulin (T6557, Sigma-Aldrich, St. Louis, MO).