Precellys 24 homogenizer

DNA was prepared employing the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s guide. 10 mg tissue was homogenized in 500 μl PBS in Precellys ceramic Kit tubes (Peqlab. Erlangen, Germany) in a Precellys 24 homogenizer (Peqlab. Erlangen, Germany) with following cycle parameters: 6000 rpm two times for 45 sec with a 60 sec break. DNA was prepared from 80 μl homogenized organs.

Kidney tissues were crushed using a Precellys 24 homogenizer (Peqlab, Erlangen, Germany) and were lysed with RIPA buffer (Sigma-Aldrich, St. Louis, MO) supplemented with protease inhibitor and phosphatase inhibitor cocktail (Thermo Scientific, Massachusetts, USA) at 4 °C for 30 min. Twenty-five microgram protein samples were separated by 10% SDS-PAGE. After semi-dry transfer, the membranes were blocked with 5% nonfat milk and incubated with primary antibody overnight at 4 °C. Then, the membranes were probed with HRP-conjugated secondary antibody for 30 min at room temperature. The protein bands were detected with ECL reagent (NcmECL Ultra, NCM Biotech). Relative band densities of the proteins were analyzed by using ImageJ software (NIH). The primary antibodies used include: anti-NLRP3 (1:800; Abcam, UK), anti-NGAL (1:1000; Abcam, UK), GAPDH were used as internal controls of renal tissues.

Whole RNA of third-instar larvae was isolated using TriFast reagent (peqlab). Tissue was homogenized using a Precellys 24 homogenizer (peqlab). Transcription to cDNA was performed using the Quantitect Reverse Transcription Kit (Quiagen). qPCR was performed with a CFX Connect cycler (biorad). A minus-RT was analyzed in a PCR for each cDNA. qPCR was performed with a CFX Connect cycler (biorad) using GoTaq SYBR Mix (Promega). Values were normalized against two housekeeping genes (actin5c and rp49) and against wild-type control (ΔΔC_q). See Table 1 for primer sequences. Each experiment was repeated at least 5 times.

Protein isolation was performed by scraping the co-cultured cells into 50 μl of lysis buffer (10 mM Tris-HCl, 10 mM Hepes, 150 mM NaCl, 5 mM EDTA, Complete EDTA-free protease inhibitor cocktail (Roche), 1 μg/ml pepstatin (Sigma), 0.5% NP40 and 1% Triton) per well, pooling 12 wells. Samples were homogenized using 1.4 mm-sized zirconium oxide beads and the Precellys 24 homogenizer (Peqlab), and protein concentrations were measured using Bradford Ultra (Expedeon) and the Infinite M200 Pro plate reader (Tecan). 20 μg of proteins were loaded on the SDS-PAGE gels. We used 4–15% gradient Mini-PROTEAN_TGX precast gels (Bio-Rad) for the detection of α-Tubulin (Neomarkers, Thermo Scientific, 1:2000), collagen (F1C3, 1:1000) and Tnc (KAF14, 1:1000). Semi-dry blotting was performed at 200 mA for 1h and subsequently at 120 mA for 1 h using the Trans-Blot SD Blotter (Bio-Rad). Blocking was performed using 1x Roti-Block (Roth) and primary antibodies were incubated overnight at 4°C. After washing with TBS-T, anti-mouse DyLight-800 and anti-rabbit DyLight-680 secondary antibodies (Cell Signaling) were used at 1:15000 and after washing, the signals were measured using the Odyssey CLx infrared imaging system (LI-COR). Quantification of 3 technical replicates was performed using the Image Studio Lite Western blot analysis software. Statistics: one-way Anova with Bonferroni post-hoc test.