Complete mini edta free protease inhibitor cocktail tablets

TurboID strains and N2 animals were synchronized by hypochlorite lysis. 40,000 synchronized L1 animals were plated to NGM seeded with concentrated OP50 food. Animals were grown at 15°C until they reached the L4 stage. Animals were then incubated overnight at 25°C, collected in M9 and washed two times in M9, once in ddH₂O and once in RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.125% SDS, 0.125% sodium deoxycholate, 1% Triton X-100 in ddH₂O). Animals were then resuspended in RIPA buffer supplemented with cOmplete mini EDTA-free Protease Inhibitor Cocktail tablets (Sigma-Aldrich). Resuspended pellets of animals were flash-frozen in liquid N₂ until further use. Worm pellets were lysed using a bead mill homogenizer. Lysate was centrifuged at 14,000× RPM. The supernatant was mixed with 80 µl Streptavidin magnetic beads (Thermo Fisher Scientific) and incubated overnight at 4°C with constant rotation. Beads were then washed for 5 min, two times with RIPA buffer, once with 1 M KCl, once with 0.1 M Na₂CO₃, and once with 2 M urea in 10 mM Tris-HCl (pH 8.0). Beads were resuspended in PBS and subjected to on-beads trypsin digestion.

As in our prior experiments (Chen et al., 2012 (link); Zheng et al., 2014 (link); Yuan et al., 2015 (link)), we adapted our standard protocols as follows “Both inner ears were rapidly removed and dissected in ice-cold PBS, pH7.4, containing complete™ mini EDTA-free protease inhibitor cocktail tablets (Sigma-Aldrich, Burlington, MA, USA, #11836170001) to remove vestibular portions. To extract total protein, tissues from two cochleae of a mouse were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich, R0278) with the cocktail proteinase inhibitors plus Phosphatase Inhibitor Cocktails II and III (Sigma-Aldrich, #P5726 and #P0044) by using a glass/glass micro–tissue grind pestle and vessel for 30 s. After 30 min on ice, tissue debris was removed by centrifugation at 15,000 × g at 4°C for 10 min and the supernatants were retained as the total protein fractions. Protein concentrations were determined using the Bio-Rad Protein Assay dye reagent (Bio-Rad, Hercules, CA, USA, #500-0114) with bovine serum albumin as a protein standard. Finally, the total protein was stored at −80°C after quantification.”

Punch biopsies were mechanically dissociated in gentleMACS M Tubes (Miltenyi Biotech, 130-093-236) using the Gentle MACS protein program. Tissue samples were processed in 500 μL of T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, 78510) and cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Sigma Aldrich, 04693159001). After dissociation, samples were centrifuged and the supernatant was collected and analyzed for protein concentration. Protein concentration was determined using the Microassay BCA Protein Assay Kit (Thermo Fisher Scientific, 23235) using BSA as the standard. Tissue lysates were analyzed with the V-PLEX Plus Human GM-CSF Kit (Mesoscale Discovery, K151RIG). Protocols were applied according to the manufacturer’s instructions and results were reported in pg/μg total protein.

All general chemicals, amino acids, bovine serum albumin, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), dimethylsulfoxide (DMSO), glucose, glucose-dependent insulinotropic polypeptide (GIP, G2269), cOmplete Mini EDTA-free Protease Inhibitor Cocktail Tablets (11836170001), and heat-inactivated fetal bovine serum (FBS; 12306C) were purchased from Sigma-Aldrich. RPMI 1640 base medium (11-875-093), antibiotic–antimycotic solutions (15240112), NP-40 Alternative (492016), Fura Red Ca²⁺ imaging dye (F3020), DiR (D12731), and agarose (BP1356-500) were purchased from Thermo Fisher. Glass-bottomed culture dishes were ordered from Mattek (P35G-0-14C). Fura Red stocks were prepared at 5 mM concentrations in DMSO, aliquoted into light-shielded tubes, and stored at −20°C until day of use (5 μM final concentration). DiR was prepared in DMSO at 2 mg/ml, aliquoted to light-shielded tubes, and stored at 4°C until use. All imaging solutions were prepared in a bicarbonate/HEPES-buffered imaging medium (formula in Table 1). Amino acids were prepared as 100×stock in the biocarbonate/HEPES-buffered imaging medium, aliquoted into 1.5 ml tubes, and frozen at −20°C until day of use. Aliquots of GIP stock were prepared at 100 μM in water and kept at −20°C until day of use.