Lysis buffer

Rat pups intestine tissue samples were isolated and suspended in Allprotect Tissue Reagent (Qiagen Inc., Valencia, CA, USA) or flash frozen in liquid nitrogen before being stored at −80°C. The frozen tissue was sectioned and suspended in lysis buffer (Cell Signaling Tech, Boston, MA, USA) containing 1 mM Phenylmethylsulfonyl fluoride (PMSF). Samples were homogenized for 3 minutes on ice. After centrifugation for 1 minute at 4°C, the supernatents were removed and stored at −80°C. To isolate proteins from cellular monolayers grown on 100 mm plates (5.5 × 10⁶ cells), the media was removed and 1 mL of PBS was added and the cells were scraped and transferred to a microfuge tube. Samples were microfuged at 5,000 rpm at 4°C for 10 minutes and the supernatents were removed. The cell pellets were resuspended in 500 μl of lysis buffer (as above). The mixture was then drawn three times through a 27-gauge needle and gently mixed on a rotating platform for 30 minutes at 4°C followed by centrifugation at 4°C for 15 minutes at 10,000 rpm. The supernatants were removed and stored at −80°C. Before use, the tissue and cellular samples were thawed on ice. A total of 5X Laemmli SDS sample buffer was added and then boiled for 3 minutes. The samples were stored at −20°C until used.

C2C12 cells were washed with ice‐cold PBS and lysed in lysis buffer (Cell Signaling Technology) supplemented with protease inhibitors (Roche, Basel, Switzerland). After centrifugation at 15,000 × g for 15 min, the protein in the supernatant was quantified using the detergent compatible protein assay kit (Bio‐Rad, Hercules, CA, USA). The quantified proteins were denatured, separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis on 4–12% gradient gels, and transferred onto a polyvinylidene difluoride membrane using the iBlot 2 Dry Blotting System (Thermo Scientific). Blots were incubated with primary antibodies in 1% BSA overnight at 4°C and then incubated with secondary antibodies in 5% skim milk at room temperature for 2 hr. The membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and visualized on a LAS‐4000 luminescent image analyzer (GE Healthcare Life Sciences, Little Chalfont, UK). Actin and GAPDH were used as loading controls.

The knee joint samples were collected 15 h after MSU i.a. injection (100 μg/10 μl/joint) and homogenized in 400 μl of lysis buffer with (Cell Signaling, Danvers, MA, United States) followed by centrifugation. The determination of phosphorylated NF-κB p65 subunit (activated) and total levels of NF-κB p65 subunit were performed using ELISA PathScan Kits (Cell Signaling, Danvers, MA, United States) according to the manufacturer’s directions. Absorbance was measured at 450 nm (Multiskan GO, Thermo Scientific) and the results are expressed as IOD ratio (total NF-κB p65/p-NF-κB p65).

HEK293 cells grown in a 15-cm dish were transfected with human GPR110-HA for 24 h. After removing the medium, cells were washed one time with PBS (pH 7.4) and resuspended in 5 mL of PBS. Cells were stimulated with 10 nM synaptamide or oleoylethanolamine (OEA) control or DMSO vesicle for 10 min followed by incubation with 1 mM DSS (i.e., adding 25 µL of freshly made 200 mM DSS in DMSO, final concentration of DMSO was 0.5%) for 30 min at room temperature (25 °C). The cross-linking reaction was quenched by addition of 150 µL of 1 M Tris-HCl (pH 7.4). The cells were lifted by gently scraping and the cell suspension was transferred to a 15-mL conical tube. Cells were pelleted by centrifugation at 1000 rpm for 10 min at 4 °C, and then lysed in 1.5 mL lysis buffer (Cell Signaling Technology) on ice for 40 min with vortexing at 5-min intervals. Cell debris was removed by centrifugation at 15,000 rcf for 15 min and the supernatants were subjected to immunopurification.

Human lung cancer cell lines H3255, and HCC827 were obtained from the Hamon Center Collection (University of Texas Southwestern Medical Center, Dallas, TX, USA) and H358, H1299, and PC9 were purchased from ATCC (Manassas, VA, USA). These cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS at 37°C in 5% CO₂. Cells were lysed with lysis buffer (Cell Signaling Technology) supplemented with 1 mM phenylmethylsulfonyl fluoride. All cell lines were checked for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit purchased from Lonza (Cat. No. LT07-118; Switzerland) and short tandem repeats profiling analysis has been submitted for authentication.