8 Quizartinib concentration and RNA was extracted according to the method of Bashyam and Tyagi [41]. 1 or 5 μg of the RNA was treated prior to qRT-PCR with RNase-Free DNase (Fermentas GmbH, St. Leon-Roth, Germany). Reverse transcription of mycobacterial

RNA was carried out using the RevertAid™ M-MuLV Reverse Transcriptase (Fermentas GmbH) and hexamers or the Access RT-PCR System (Promega, Mannheim, Germany) according to the manufacturer’s protocols. The porin cDNA from M. smegmatis SMR5 [42] and M. fortuitum was quantified either by amplifying a fragment of about 100 bp using the primers (mspATaqfw, mspATaqbw, mfpqPCRfw and mfpqPCRrev) as well as TaqMan-probes (mspATaqProbe and mfpqPCRprobe) or the primers porM1-51-sybr-fw and bw based on SYBR Green detection chemistry (Table 1). The qPCR reactions were performed using the SensiMix DNA Kit (Quantace Ltd., Berlin, Germany) or the Access RT-PCR System (Promega) according to the manufacturer’s protocol. TaqMan quantification was carried out by running a first step at 95°C for 10 min followed by 40 cycles with 30 s at 95°C and 1 min at 58°C. SYBR Green quantification was performed by initial 10 min at 95°C followed by 40 cycles with 15 s at 95°C, 10 s at 58°C and 20 s at 72°C. Afterwards, the amplicon’s melting temperature was determined ramping the temperature from 60°C to 90°C by 0.5°C steps and acquiring the fluorescence signal. cDNA amounts were determined

by three measurements for each sample using a calibration curve established with known amounts of linearised pSSa100 [13] in case of M. smegmatis or pSSp107 in case of M. fortuitum. DNase treated and non-reverse-transcribed I-BET-762 supplier controls were performed with the same samples to guarantee the absence of contaminating genomic DNA. In addition to the qRT-PCR experiments, the amount of porin in isolates of M. fortuitum and M. smegmatis was determined by Enzyme-Linked Immunosorbent Assay (ELISA). Protein was isolated from mycobacteria using the detergent nOPOE as described above. The isolated protein (15 Ureohydrolase μl corresponding approximately to 25 μg) was diluted in 50 mM NaHCO3, pH 9.6 to yield a protein concentration of 1 μg/100 μl. Aliquots (100 μl) of the sample

and dilutions thereof were loaded to wells of a Nunc-Immuno Maxisorp Module (Nalgene Nunc International, NY, USA). After incubating the samples at 4°C overnight, wells were washed twice with TBS-T (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM MgCl2 and 0.05% Tween 80). The surface was blocked with 3% powdered skim milk in TBS for 1.5 h at room temperature followed by three steps of washing with TBS-T. Samples were then treated with the primary antibody for 1.5 h at room temperature, using a 1:1500 dilution of the antiserum pAK MspA#813 [8] in TBS. The wells were washed five times with TBS-T and were incubated for 1 h at room temperature with a 1:7500 dilution of Peroxidase-conjugated AffiniPure F (ab’) 2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson Immuno Research, Soham, UK) in TBS.