Fastprep fp120 instrument

Cerebella of P7 and P30 Cstb^−/− and control mice (n = 3 per genotype) were lysed with 50 mM Tris (pH 8.0), 0.5% Nonidet P-40, 10% glycerol, 0.1 mM EDTA, 250 mM NaCl, 0.1 mM Na₃VO₄, 50 mM NaF, 4 mM dithiothreitol (DTT), 1× Protein inhibitor cocktail (Roche, Basel, Switzerland) using Lysing Matrix D tubes (Qbiogene, Carlsbad, CA, USA) and FastPrep® FP120 Instrument (Qbiogene, Carlsbad, CA, USA). Lysed proteins (15 µg) were separated with Protean TGX precast gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred on the nitrocellulose membrane. The primary antibodies used were rabbit anti-rat GABRA6 (1∶1000) (Synaptic Systems, Göttingen, Germany) and mouse anti-rat β-tubulin (1∶10 000) (Sigma, St. Louis, MO, USA), and the secondary antibodies used were anti-rabbit-IRDye 800CW (1∶10 000) (LI-COR Biosciences, Lincoln, NE, USA) and anti-mouse-Alexa Fluor 680 (1∶10 000) (Invitrogen®, Life Technologies, Carlsbad, CA, USA). The bands were detected with Odyssey infrared reader (LI-COR Biosciences, Lincoln, NE, USA). Signal intensities were detected with Image Studio 3.1 (LI-COR Biosciences, Lincoln, NE, USA) and normalized to the intensity of β-tubulin.

To isolate the RNA for quantitative real-time PCR (RT-qPCR) analysis, all P. aeruginosa strains were grown at 37°C overnight in brain–heart infusion broth (BHI), diluted 100-fold in fresh 10 ml BHI in a 50-ml conical tube (BD Biosciences), and incubated at 37°C with shaking at 250 rpm for 6 h. The bacteria were harvested and disrupted mechanically (Fast Prep FP120 instrument; Qbio-gene). The RNeasy Mini Kit (Qiagen) was used for the subsequent RNA purification. RNA concentration and purity were determined by UV absorption at 260 and 280 nm.

For DNA isolation, 5 g of sample material was physically disrupted with a sterile pestle and mortar and resuspended in 10 ml of 0.85% NaCl. Total community DNA was extracted from 2-ml aliquots after centrifugation (16,750 × g for 20 min at 4°C) using the FastDNA Spin kit for soil (MP Biomedical, Solon, OH). In a deviation from the manufacturer’s protocol, pellets were homogenized twice in a FastPrep FP120 instrument (Qbiogene, Inc., Bio 101, Carlsbad, CA) for 30 s at speed 5.0 m·s⁻¹.

Sampling was carried out 2 weeks after planting (young plants) for treatments with and without FZB42 application, followed by a second sampling 4 weeks after planting (mature plants) for control and co-inoculated treatments with FZB42 and R. solani. The total community DNA was extracted per treatment, and habitat from two young plants and three mature plants (two independent DNA extractions were performed for each plant and the DNA was pooled prior to PCR), according to Bragina et al. (2011 (link)). Briefly, 5 g of plant material were physically disrupted with sterile pestle and mortar and resuspended in 10 ml of 0.85% NaCl. Two ml of suspension were centrifuged (16,500 × g, 20 min, 4°C) and the obtained pellets were used for isolation of the total-community DNA with the FastDNA® SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA). For mechanical lysis, the cells were homogenized twice in a FastPrep® FP120 Instrument (Qbiogene, BIO101, Carlsbad, CA, USA) for 30 s at a speed of 5.0 m s⁻¹ and treated according to the manufacturer's protocol.