Of protease and phosphatase inhibitors

Total protein lysates from cells were extracted by using the RIPA solution (Beyotime, China) with a cocktail of protease and phosphatase inhibitors (Roche), followed with incubation on ice for 30 min and centrifugation at 12,000 × g for 15 min at 4 °C. The final protein concentration of each sample was determined by a BCA kit (Thermo Scientific).
The protein expression level was detected by Western blotting analysis. The supernatants (20 μg total protein) from protein lysates were subjected to SDS-PAGE gel according to standard procedures in Bio-Rad system. Mouse anti-endophilin A2 antibody, rabbit anti-Caveolin-1and rabbit anti-GAPDH were used as the primary antibody. The signal was further detected by using the secondary antibody of goat anti-rabbit IgG or goat anti-mouse IgG. The intensity of specific binding bands was calculated against the endogenous control (GAPDH), and data were showed as fold change against the control.

Upon extensive perfusion of whole hearts from WT and frataxin KO mice with ice-cold PBS, LV tissue was excised and homogenized in 500 μL of ice-cold RIPA buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS and 1x solution of protease and phosphatase inhibitors (Roche Diagnostics)] using a Dounce glass homogenizer [12 (link)]. Immunoprecipitation was performed as described previously [12 (link)], but using Pgc1α antibody (5 μg; ab54481; Abcam).