Ab37117

The intestinal tissues (20 mg) was homogenized with 200 μL RIPA lysis containing proteinase inhibitors, and then centrifuged at 12000 rpm for 5 minutes for the collection of supernatant containing protein for further analysis. The concentrations of protein samples were determined using BCA kit (Beijing Puli Gene Technology Co., Ltd. Beijing, China). Protein samples were loaded on a 10% SDS-PAGE gel and transferred to PVDF membrane. After blocked in 5% nonfat milk for 1 hour, the membranes were incubated with the primary antibodies anti-ATF6α (ab203119, 1:1000, Abcam Inc., Cambridge, MA), anti- PERK (70R-17036, 1:1000, Fitzgerald Inc, MA) and anti-IRE1α (ab37117, 1:1000, Abcam Inc., Cambridge, MA) at 4 °C overnight. After washed three times with TBS-Tween (TBS-T), the membranes were incubated with appropriate HRP-conjugated secondary antibodies (dilution 1: 5000) at room temperature for 1 hour. Blots were developed with chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) and imaged using ImageQuant™ LAS 4000 (General Electric Company, Boston, USA). The relative band density was qualified using Quantity One v4.6.2 software.

EC109 cells were seeded into six-well plates and were treated with different agents for 24 h when the confluence reached 80%. The cells were washed with PBS and lysed in 100 μl radioimmunoprecipitation assay (RIPA). After brief sonication and centrifugation, the supernatants were collected for protein concentration measurement by bicinchoninic acid (BCA) kit. The proteins in each group were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. After blocking with 5% milk, the membranes were incubated with primary antibodies (1:1,000), including anti-GRP78 (ab21685, Abcam), antiactivating transcription factor 6 (ATF6) (ab203119, Abcam), anti-inositol-requiring enzyme 1α (IRE1α) (ab37117, Abcam), antiprotein kinase RNA-like endoplasmic reticulum kinase (PERK) (70R-17036, Fitzgerald), and anti-β-actin (E2317, Cell Signaling Technology). After incubation overnight at 4°C, the membranes were washed and incubated with secondary antibodies (diluted 1: 2000) at room temperature for 1 h. Enhanced chemiluminescence (ECL) was used for signal development. BioRad imaging system was used to capture the chemiluminescence. Analysis was conducted using Quantity One software, and the relative protein levels were expressed as the intensity ratios of target protein to β-actin.