Ripa buffer

For whole cell protein extraction, isolated rat cardiomyocytes were lysed in RIPA buffer (Cayman #10010263) supplemented with protease and phosphatase Inhibitor cocktail (Cell Signaling #5872S) on ice for 1 h. The supernatant was collected and combined with 4X loading dye (Li-COR #928-40004), supplemented with 10% 2-mercaptoethanol, and boiled for 10 min. The resulting lysate was resolved on SDS-PAGE gel and protein was blotted to nitrocellulose membrane (Li-COR #926-31902) with a mini Trans-Blot Cell (Bio-Rad). Membranes were blocked for an hour in Odyssey Blocking Buffer (TBS) (LI-COR #927-50000) and probed with corresponding primary antibodies overnight at 4 °C. Membranes were rinsed with TBS containing 0.5% Tween 20 (TBST) three times and incubated with secondary antibodies TBS supplemented with extra 0.2% Tween 20 for 1 h at room temperature. Membranes were washed again with TBST (0.5% Tween 20) and imaged on an Odyssey Imager. Image analysis was performed using Image Studio Lite software (LI-COR). All samples were run in duplicates and analyzed in reference to GAPDH.

Streptozotocin (STZ) and protease inhibitor cocktail (PIC) were purchased from Sigma‐Aldrich Co., LLC. (Missouri, USA). Isoflurane, 10% formalin neutral buffer solution (pH 7.4), and liquid paraffin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Hair‐removing body cream was purchased from Kracie Holdings., Ltd. (Tokyo, Japan). RIPA buffer was purchased from Cayman Chemical Company (Michigan, USA). Edaravone (Radicut®) was purchased from Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). Pregabalin (Lyrica®) was purchased from Pfizer Inc (Tokyo, Japan). SMTP‐44D was generously donated by TMS Co., Ltd. (Tokyo, Japan). The structure of SMTP‐44D is shown in Figure 1.

Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA solution, and protease inhibitor cocktail (PIC) were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). RIPA buffer, leukotriene B4-d4 (LTB4-d4), (±)11(12)-EET (11(12)-EET), (±)11(12)-DiHET (11(12)-DHET), (±)14(15)-EET (14(15)-EET), and (±)14(15)-DiHET (14(15)-DHET) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Four percent paraformaldehyde phosphate-buffered solution, trisodium citrate, formic acid (abt.99%) for HPLC, ultrapure water for LC/MS, 2-propanol for LC/MS, acetonitrile for LC/MS, and ethyl acetate were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Triton^® X-100 was purchased from MP BIOMEDICALS. (Santa Ana, CA, USA). Cellstain^®-DAPI solution was purchased from Dojindo Laboratories (Kumamoto, Japan). SMTP-44D [9 (link)] was generously donated by TMS Co., Ltd. (Tokyo, Japan). The structure of SMTP-44D is shown in Figure 5.

Acetaminophen (Spectrum Chemical Corp, New Brunswick, NJ, #AC100), triethanolamine (Sigma-Aldrich Corp, # t58300), 2-vinylpyridine (Alfa Aesar, Ward Hill, MA, #A14056), RIPA Buffer (Cayman Chemical Co., Ann Arbor, MI, # 10010263), GSH MES Buffer (Cayman Chemical Co., # 703010), cOmplete Protease Inhibitor Cocktail (Roche, Penzberg, Germany, #4693124001).

Frozen aliquoted cardiac tissue obtained from similar locations of the heart was pulverized finely using a liquid nitrogen-cooled mortar and pestle. 1x Radioimmunoprecipitation assay (RIPA) buffer (Cayman Chemical Company: 10010263) supplemented with 1x protease inhibitor cocktail (Cell Signaling Technology: 5872S) and 1:200 diluted endonuclease (Lucigen: OC7850K) was immediately added to the pulverized tissue at a constant ratio of 15 μL/mg of tissue. The sample was then mechanically homogenized using a handheld homogenizer until visible chunks of tissues were dissociated. The sample was incubated for 10 min on ice to allow endonuclease to cleave DNA. After processing of all samples, the samples underwent two freeze-thaw cycles, after which, equal-volume of 5% SDS-10% glycerol boiling (SGB) buffer was added to each sample. The samples were vortexed thoroughly then heated to 100°C for 8 min. Residual undissolved cell debris were removed from the resulting samples by centrifugation at 8000 g for 5 min at room temperature (22°C). The concentrations of the total protein were determined using Bicinchoninic acid (BCA) assay; all samples were diluted to 4 μg/μL using RIPA:SGB buffer. The diluted total protein lysates were aliquoted and stored at −80°C until further processing.