Bullet blender 24

All mouse studies were performed following protocols that received approval from the Indian Institute of Science Education and Research Pune Institutional Animal Ethics Committee. The mice were genotyped using an established protocol (5 (link)). The mouse brain membrane proteomes were prepared using a previously described procedure (15 (link)). Briefly, the mice were first anesthetized with isoflurane and euthanized by cervical dislocation, following which the brains of the mice were harvested. A fresh half brain was suspended in 500 μl of cold sterile DPBS and homogenized using a tissue homogenizer (Bullet Blender 24, Next Advance) with one scoop of glass beads (0.5-mm diameter; Next Advance) at a speed setting of 8 for 3 min at 4 °C. To the brain homogenate, an additional 500 μl of cold sterile DPBS was added, mixed by pipetting, and centrifuged at 1000 × g for 5 min at 4 °C to separate the tissue debris. The resulting supernatant (∼700 μl) was separated and centrifuged at 100,000 × g for 45 min at 4 °C. The resulting supernatant was discarded, and the pellet (membrane proteome) was washed with cold sterile DPBS (three times) and resuspended in 1 ml of cold sterile DPBS by pipetting. The protein concentration of the brain membrane proteome was estimated using BCA protein assay kit (Pierce).

ATP was extracted from 10 μg of flash frozen cardiac muscle. Ice cold 0.4 M perchloric acid was added to the cardiac tissue and it was immediately homogenized using the Bullet Blender 24 (Next Advance) at 4°C. The reaction was neutralized with ice cold 2 M KHCO₃ [58 ]. The extracted ATP was used with an ATP Bioluminescent Assay Kit (Sigma) according to the manufacturer's protocol and measured using Wallac Victor2, 1420 Multilabel Counter (PerkinElmer) [37 (link)].

The whole pituitaries were homogenized with a bead method using Bullet Blender24 (Next, Advance, Troy, MI, USA). The RNA was isolated by a PureLink™ RNA Mini Kit (Ambion, Cambridge, UK) according to the manufacturer’s recommendations. The concentration and quality of the RNA were estimated using TapeStation2200 (Agilent, Palo Alto, CA, USA). The RIN parameter values for all the RNA samples were over 7. The TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA) was used for cDNA library preparation, with 300 ng of RNA as the input amount according to the supplied protocol. The reverse transcription during the procedure was non-strand-specific. Flowcell clustering was performed using the TruSeq SR Cluster Kit v3-cBot HS (Illumina, San Diego, CA, USA). The sequencing was conducted on a HiScanSQ System (Illumina, San Diego, CA, USA) in single 85 bp cycles (four technical replicates) using TruSeq Kit v3-HS chemistry (Illumina, San Diego, CA, USA), as described by Piórkowska et al. [16 (link)].

In each sample, an appropriate volume of culture was drawn to give ~4 x 10⁶ cells for RNA isolation. Cells were centrifuged at 5,000 g for 5 min, and the supernatant was removed. Microalgae cells were homogenized using stainless steel beads (0.2 mm, Next Advance) for 3 min at speed 10 in a Bullet Blender^® 24 (Next Advance). Total RNA was extracted using a NucleoSpin^® Plant II kit (Macherey-Nagel) following the manufacturer’s instructions. The extracted RNA was treated twice with DNase I to avoid further PCR amplification of residual traces of genomic DNA. RNA samples were quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and the quality was checked in agarose gels.
Total RNA (1 μg) from each sample was reverse-transcribed using an iScript™ cDNA Synthesis kit (Bio-Rad) in a reaction volume of 20 μL according to the manufacturer’s protocol. All cDNA reactions were finally diluted 10-fold by adding 180 μL of nuclease-free water. The absence of genomic DNA contamination was confirmed by direct PCR amplification of two randomly selected RNA samples in the absence of cDNA synthesis.

Fibroblasts expressing WT or mutant UGDH were assayed for UGDH-specific activity essentially as previously described^{56 (link)}. Fibroblasts were cultured in 15 cm plates, washed three times with cold 1x PBS, and centrifuged at 1500 rpm for 5 min. Cells were resuspended in twice the pellet volume of Lysis Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail). Samples were transferred to tubes with an equal volume of acid washed glass beads (Sigma) and lysed in the Bullet Blender 24 (Next Advance) at speed 8 for 3 min. The resulting supernatant was centrifuged at 13,000 rpm for 15 min to obtain final lysates. Enzymatic activity of the lysates (50 µg) was assayed with 1 mM UDP-glucose and 1 mM NAD⁺ in the presence or absence of 1 mM UDP-xylose, a UGDH-specific inhibitor, and monitored for changes in NADH, A₃₄₀. Reaction rates for samples containing UDP-Xyl were subtracted from samples without UDP-Xyl to obtain UGDH-specific activity reported as [NADH] in nmol min⁻¹ mg⁻¹ lysate as described above. Each fibroblast cell line analyzed contained three or more technical replicates for reactions with and without UDP-Xyl plotted as mean ± SD. Statistical significance was assessed by one-way ANOVA (Prism).

Lungs were separated by lobes into RINO tubes (cat no: NAVYR1-RNA; Next Advance) and filled with ice cold milliQ water. Lungs were then homogenized using a bullet blender (Bullet Blender 24; Next Advance) at max speed for 1 minute at 4°C followed by placing samples on ice for a 2-minute cool down, then repeat the process for 2-4 more cycles. Samples were centrifuged and inhibitors were added to maintain sample integrity: 1X Halt (1:100, cat no: 78442, Thermo Fisher Scientific), 1X Z-PUGNAc (1:1000, cat no: 3384, Tocris), 1X Thiamet G (1:1000, cat no: 13237; Cayman Chemical) and 1X Benzonase (1:1000, E1014; Millapore Sigma). Homogenized lungs were split for protein, RNA, and hydroxyproline assays.