Nupage precast 4 12

Total proteins from C‐MSC were obtained by Laemmli lysis buffer. After quantification with DC protein assay (Bio‐Rad, Hercules, California, USA), proteins were run on SDS–PAGE gel (NUpage precast 4–12%; Invitrogen, Carlsbad, California, USA) and transferred to nitrocellulose membrane (Bio‐Rad, Hercules, California, USA). The membrane was blocked in 5% skimmed milk‐TBS for 1 h at RT and incubated overnight at 4°C with primary antibodies against GAPDH, PPARγ, and CD36 (see Appendix Table S7). After washes, the membrane was incubated for 1 h at RT with the appropriate HRP‐conjugated secondary antibody goat anti‐rabbit or goat anti‐mouse (GE Healthcare, Chicago, Illinois, USA). Blots were washed and developed with the ECL system (Bio‐Rad, Hercules, California, USA). Images were acquired with the Alliance Mini 2 M System (UVITEC, Cambridge, UK), and densitometric analysis was performed using Alliance Mini4 16.07 software (UVITEC, Cambridge, UK). Data are normalized expressing as 1 the comparison group in order to highlight the fold differences between different groups or treatment.

Total proteins from murine total heart tissue lysates were obtained by Laemmli lysis buffer. After quantification with DC protein assay (Bio‐Rad, Hercules, California, USA), proteins were run on SDS–PAGE gel (NUpage precast 4–12%; Invitrogen, Carlsbad, California, USA) and transferred to nitrocellulose membrane (Bio‐Rad, Hercules, California, USA). The membrane was blocked in 5% skimmed milk‐TBS for 1 h at RT and incubated overnight at 4°C with primary antibodies against GAPDH, PPARγ, MDA, and CD36 (see Appendix Table S7). After washes, the membrane was incubated for 1 h at RT with HRP‐conjugated secondary antibody goat anti‐rabbit (GE Healthcare, Chicago, Illinois, USA). Blots were washed and developed with the ECL system (Bio‐Rad, Hercules, California, USA). Images were acquired with the Alliance Mini 2 M System (UVITEC, Cambridge, UK), and densitometric analysis was performed using Alliance Mini4 16.07 software (UVITEC, Cambridge, UK). Data are normalized expressing as 1 the comparison group in order to highlight the fold differences between different groups or treatment.

Total proteins from LV and RV C-MSC were obtained by Cell Lysis Buffer (Cell Signaling). After quantification with DC protein assay (Bio-Rad), proteins were run on SDS-PAGE gel (NuPAGE precast 4–12%, Invitrogen) and transferred to the Trans-Blot^® Turbo^TM nitrocellulose membrane (Bio-Rad) with the Trans-Blot^® Turbo^TM transfer system. The membrane was blocked in PBS containing 0.05% Tween^® 20 (Sigma–Aldrich) and 5% skimmed milk (ChemCruz) for 1 h at RT and incubated overnight at 4°C with the primary antibodies against GAPDH and the main adipogenic proteins PPARγ, PLIN1, and FABP4 (see Supplementary Table S2). After washes in PBS containing 0.05% Tween^® 20 (Sigma–Aldrich), the membranes were incubated 1 h at RT with the appropriate HRP-conjugated secondary antibody (see Supplementary Table S3). Blots were washed and developed with the ECL system (Amersham) and images acquired and quantified with the UVItec Cambridge system. The normalization was performed on the housekeeping protein GAPDH.