Rnase inhibitor

The sedimentation of mitoribosomes was analyzed using sucrose density gradients. The isolated mitochondria were solubilized with the lysis buffer (10 mM Tris–HCl, pH 7.4, 50 mM KCl, 20 mM MgCl₂, 1% Triton X-100, 1× protease inhibitor cocktail (Roche), 40 U/μl RNase inhibitor (Agilent). 700 μg of protein was loaded onto a linear 10–30% gradient containing 10 mM Tris–HCl, 100 mM KCl and 20 mM MgCl₂ supplemented with 1x protease inhibitor cocktail (Roche) and centrifuged at 79 000 g for 15 h at 4°C (Beckman Coulter; SW41 rotor). The gradients were collected from the top into 20 fractions of 450 μl/fraction, and fractions 1–18 were analyzed by immunoblotting, with fractions 1 and 2 pooled together.

2.5 µg total RNA of each spleen was subjected to a reverse transcription reaction in a total volume of 40 µl employing 5 × FS-Buffer, 3 µg random hexamer primer, 200 U M-MLV transcriptase, 0.02 µmol dNTPs, and 0.4 µmol DTT (all Invitrogen^TM GmbH) as well as 40 U RNAse inhibitor (Agilent Technologies). 70 °C denaturation step was followed by a 1.5 h 42 °C reverse transcription step. 1 µl of individual cDNA preparations from each IL-10R-blocked and isotype-treated mouse, respectively, was pooled. The two pools were diluted 1:200 for subsequent qPCR which was performed as described previously^{47 (link)}. Transcripts of three housekeeping genes and 32 genes involved in cytokine-, interferon-, chemokine- and innate immunity-related signaling were quantified as fold changes using the ΔΔC_T-method. For a detailed list of genes and primer sequences, see supplemental Table S2. Transcripts reaching a threshold fold change of 1.5 were regarded as potentially regulated and subjected to RT-qPCR in non-pooled samples.