Genetools program

Aβ was quantified using a dot blot assay [36 (link)]. Briefly, brain protein extracts (1 µL) were spotted onto the nitrocellulose membranes and dried (37 °C, 1 h). The membranes were washed with TBST buffer (RT, 15 min) and blocked with 5% BSA in TBST buffer (RT, 1 h). Blocked membranes were washed three times with TBST buffer (10 min each) and incubated with monoclonal anti-Aβ antibody (CS #8243; 4 °C, 16 h). Membranes were then washed three times with TBST buffer (10 min each) and incubated with goat horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody. Positive signals were detected using Western Bright Quantum-Advansta K12042-D20 and GeneGnome XRQ NPC chemiluminescence detection system. Signal intensity was assessed using the Gene Tools program from Syngene.

Cell pellets were lysed with Cell Lytic M reagent (Sigma, MI, USA)) containing a protease inhibitor cocktail (Sigma, MI, USA). Proteins in crude lysates were quantified using the bicinchoninic acid assay (BCA) Protein Assay (Pierce Biotechnology, MA, USA)). A total of 20 μg of whole-cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Merck Millipore, Madrid, Spain). Blots were probed using primary antibodies (Table S3). Proteins were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-mouse (P0447, Dako, Barcelona, Spain) or anti-rabbit (P0217, Dako, Barcelona, Spain), incubated for 1 h at room temperature. Band intensity on the blots was quantified using the GeneTools Program (SynGene, Cambridge, UK).

The T-RFLP analysis of bacterial and methanogenic communities using FAM-labeled PCR products was done as described previously (Sträuber et al., 2012 (link); Lucas et al., 2015 (link)). PCR product quality was checked by agarose gel electrophoresis and amplicons were purified with SureClean (Bioline, Luckenwalde, Germany). Purified PCR products were quantified after electrophoresis in 1.5% agarose gels with ethidium bromide staining using the GeneTools program (Syngene, Cambridge, UK). The purified PCR products were digested with restriction endonucleases purchased from New England Biolabs (Schwalbach, Germany). The mcrA amplicons were digested with MwoI and the 16S rRNA amplicons with RsaI, using 2 units of the respective enzyme for digesting 10 ng of PCR product at 37°C overnight. The subsequent T-RFLP analysis was done for the mcrA amplicons with the GeneScan^TM-500Rox^TM (Applied Biosystems, USA) as fragment size standard and for the 16S rRNA amplicons with the MapMarker1000 (BioVentures Inc., USA). Resulting electropherograms were analyzed by using the GeneMapper 5 software (Applied Biosystems) and processed according to Abdo et al. (2006) (link). To differentiate between peaks and background, signals with low peak areas were removed according to eight times the standard deviation.

Equal amounts of total protein extracts (lysis buffer: SDS 1%, Tris pH 7.4 10mM, Sodium orthovanadate 1 mM). were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF Transfer membrane (Thermo Scientific, Illkirch, France). Antibodies used for western blotting were YAP1 (Proteintech, Manchester, UK), anti-Flag (Sigma Aldrich), HA-tag (Cell signaling), β-actin (Cell signaling), anti-mouse IgG-HRP (Santa Cruz Biotechnology, CA, USA), anti-rabbit IgG-HRP (Santa Cruz). Antibody binding was visualized with the enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, Illkirch, France). For quantification, luminescence was detected with a Charge Couple Device (CCD) camera and analyzed using the GeneTools program (Syngene, Cambridge, UK).The original western blot figures can be found in supplemental materials (Figure S6).

Western blots were imaged and their relative intensities were quantified using the ChemiGenius Bio-Imaging System with the GeneTools program (SynGene). Relative protein levels were normalized to the housekeeping protein GAPDH while mRNA levels were normalized to α-tubulin. Data were plotted as means ± SEM and tested for significance with Student's t-test (two data points) or a one-way analysis of variance (three or more data points; Holm-Sidak test) to assess significant difference, with p < 0.05 accepted as statistically significant.