Phosstop inhibitor cocktail

Cells were seeded onto 10 cm plates at a density of 1.0 × 10⁶–1.5 × 10⁶ cells. Following experimental treatments, plates were scraped in DPBS containing protease and phosphatase inhibitors (0.5 mM PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 mM Na₃VO₄). Cells were pelleted and resuspended in RIPA lysis buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) containing a Roche PhosSTOP inhibitor cocktail in addition to the aforementioned protease and phosphatase inhibitors.

Immediately after ex vivo analysis, RV tissue was then washed in ice‐cold saline and snap‐frozen. RV tissue was then homogenized using an Omni international tissue grinder (Thermo Fisher Scientific, Waltham, MA) in ice‐cold RIPA lysis buffer (Thermo Fisher) containing proteinase inhibitor cocktail (EMD Millipore/Sigma‐Aldrich, St. Louis, MO) and PhosStop inhibitor cocktail (Roche, Indianapolis, IN). After homogenization, lysate was sonicated for ten one‐second pulses at 100% power and then centrifuged. The supernatant was saved and used as whole lung lysate. Protein concentration was measured using BCA Protein Assay (Thermo Fisher). Rabbit polyclonal anti‐phospho‐p38MAPK, anti‐total p38MAPK, anti‐bcl2, and anti‐bax (all used at 1:1000, and from Cell Signaling, Danvers, MA) and mouse monoclonal anti‐Vinculin loading control (1:5000; Calbiochem; Billerica, MA) primary antibodies were used, all diluted in Pierce Protein‐Free T20 Blocking Buffer (Thermo Fisher). All antibodies used have been extensively validated (Lin et al. 2005; Bernal‐Mizrachi et al. 2006; Bikkavilli et al. 2008; Tang et al. 2008; Slone et al. 2011; Choi et al. 2016; Jiang et al. 2016; Zhang et al. 2017). Rabbit‐HRP (Cell Signaling) and mouse‐HRP (KPL, Gaithersburg, MD) secondary antibodies were diluted 1:2000 in Pierce Protein‐Free T20 Blocking Buffer (Thermo Fisher). Densitometry was performed using ImageJ.

Mouse pancreatic islets were isolated by collagenase digestion as previously described (44 (link)) from 15-week-old female GIRKO mice given HFD for 4 weeks. After isolation, islets were allowed to recover overnight. Fifty handpicked islets per mouse were loaded into the Biorep Perifusion System with each chamber containing islets belonging to unique individuals. Islets were perifused at a rate of 120 μl/min with Krebs buffer containing 2.8 mmol/L glucose for 20 min, 16.7 mmol/L glucose for 30 min, followed by washout for 6 min, and chased for 20 min with 30 mM potassium chloride as a depolarizing stimulus. Recovered islets were lysed by shearing with a 26 1/2G sterile needle in lysis buffer containing 1% NP-40, 0.05% deoxycholate, 0.1% SDS, 0.2% Sarkosyl, 10% Glycerol, 1 mM DTT, 1 mM EDTA, 10 mM NaF, 50 mM Tris (pH 8.0), 1× Complete protease inhibitor cocktail (Roche), and 1× PhosStop inhibitor cocktail (Roche) in PBS. Secreted insulin was measured using ELISA (Mercodia), and results were normalized to total insulin content in islet lysates.