T10 basic ultra turrax

The Zn²⁺: GnRH [6-D-Phe] oil depot formulations preparation was performed under a laminar flow cabinet in a two-step process. The gelling agent Aluminiumdistearte Alugel 30 ®HEP (AlSt) was weighed and suspended in the oil vehicle miglyol 812 (MCT) to a final weight of 95 g (corresponding to 100 mL). The prepared mixture was heated at 174 °C for 2 hours under N₂. After cooling down to 25 °C the wetting agent, Phospholipon 90 H (PL 90 H), and the resuspendibility enhancer, Kolliphor ELP (KL ELP), were incorporated into the gelled oil matrix and stirred at 160 °C for 1 hour under N_2. The castor oil: MCT 50:50% (w/w) matrix was heated and agitated under inert atmosphere (N₂) at 60 °C and then cooled down to room temperature at 25 °C. The cryo-ground Zn²⁺: GnRH [6-D-Phe] lyophilizate was suspended in the prepared oil matrices at ambient temperature of 25 °C using an Ultra-Turrax T-10 basic (IKA Labortechnik, Germany) for 5 minutes at 2000 rpm. 139.8 mg of Tris buffer salt consisting of 2.01 g Trizma HCl and 1.485 g Trizma Base was added to the in-situ complex formulation. The 5 mg/mL Zn²⁺: GnRH [6-D-Phe] oil depot suspensions were aliquoted in 20 R glass vials. The prepared suspensions were stored at 2–8 °C.

A total of 40 mg of untreated and treated betulin samples were dispersed in 100 mL sterilized deionized water with a rotor homogenizer Ultra-Turrax T10 basic (IKA Labortechnik, Staufen, Germany) for 4 min at 11,500 rpm and then with an ultrasonic homogenizer Hielscher UP200Ht (Hielscher Ultrasonics, Teltow, Germany) for 2.5 min at 20% amplitude, 150 W power and 60% continuance. After intermediate cooling for 5 min, homogenization with ultrasonic homogenizer was repeated for another 2.5 min.
Both betulin samples were sterilized by autoclaving before preparation of dispersions. Autoclaving treatment was performed at 121 °C for 20 min using 3870ELV-D autoclave sterilizer (Tuttnauer, Breda, Netherlands).

Whole hearts were homogenized in 500 μl ice-cold lysis buffer (25 mmol/L Tris HCl, ph 7.4, 5 mmol/L MgCl₂, 10% glycerol, 100 mmol/L KCl, 1% NP40, 0.3 mmol/L dithiothreitol, 5μl protease/phosphatase inhibitor cocktail, 5 mmol/L nicotinamide, 1 μmol/L orthovanadate, 50 mmol/L sodium fluoride, 1 mmol/L sodium butyrate and 5 mmol/L sodium pyrophosphate), sonicated for 1 min using an Ultra-Turrax T10 basic (IKA Labortechnik, Staufen, Germany), and separated by centrifugation (10 000 x g for 30 min at 4°C). 500 μg of protein were incubated with 50 μl beads (1.5 mg) and 5 μg anti-PGC-1α antibody (Santa Cruz, Heidelberg, Germany), rotating overnight at 4°C. Beads were collected using a magnetic rack, washed with washing-buffer (Immunoprecipitation kit, Invitrogen, Carlsbad, CA), dissolved in loading buffer, and boiled. The immunoprecipitates were then separated by SDS-PAGE and immunoblotted using anti-PGC-1α-antibody (1:200; Santa Cruz, Heidelberg, Germany) and subsequently with acetyl-lysine antibody (1:1.000; Cell Signaling, Boston, MA).