Bca kit

As previously described [22 (link)], cells were washed twice with PBS and solubilized with radio immunoprecipitation assay (RIPA) buffer (Vazyme Biotec Co., LTD, Nanjing, China) supplemented with protease inhibitor (Sigma). Lysates were quantitated with bicinchoninic acid (BCA) kit (Vazyme Biotec Co., LTD); equivalent amounts of protein were subjected to electrophoresis, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Blocking with 5% dried skimmed milk in TBST (Tris Buffered Saline and 0.1% Tween 20) for 2 h at room temperature, the membranes were incubated with different primary antibodies, including KLF13 (#41724, SAB, Maryland, MD, USA), FASN (sc-20140 AC, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), FABP4 (sc-18661, Santa Cruz Biotechnology, Inc.), PPARγ (#2435, CST, Cell Signaling Technology, Inc., Danvers, MA, USA), and β-tubulin (KM9003, Sungene, Tianjin, China), at 4 °C overnight. After washing, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and developed with ChemiDoc^TM XRS + Chem iluminescence detection system (Bio-Rad, Hercules, CA, USA). Image Lab5.2 was used for densitometric analysis of the expressed protein bands.

Cells were washed in precold PBS for 3 times before cell lysis for 30 min at ice with 100 μL/50 mL cell lysate. Then, the cell lysis was centrifuged at 4°C and 12000 rpm for 10 min with supernatant collected. BCA kit (Vazyme, Nanjing, China) was used to detect the concentration of proteins. The proteins (loading volume of total cells 80 μg, sediments of 30 μL; the order was recorded) were separated on a 10% SDS-polyacrylamide gel and transferred into PVDF membrane (Millipore, Billerica, MA) to terminate the unspecific reaction with 5% skim milk powder for 1 h. After that, the membranes were incubated with rabbit anti human KDM1A (2139S, 1 : 1000, Cell Signaling Technology, Boston, USA), rabbit anti human DACT1 (ab42547, 1 : 1000, Abcam, Cambridge, USA), and rabbit anti human Anti-Histone H3 (acetyl K27, ab177178, 1 : 1000, Abcam, Cambridge, USA) for overnight at 4°C. The membranes were then washed with TBST for 3 × 10 min before incubation with horseradish peroxidase labeled goat anti rabbit IgG (1 : 5000, CoWin Biosciences, Beijing, China) at room temperature for 1 h. After TBST washing for 3 × 10 min, the membranes were analyzed using a chemiluminescence imaging system (Tannon, Shanghai, China). GAPDH was used as an internal control.

A total of 30 islets/well were seeded in a 96-well plate, and MIN6 cells were seeded in a 48-well plate. Then, the cells were transfected with circGlis3 overexpression plasmid or circGlis3 knockdown plasmid using Lipo2000 in accordance with the manufacturer’s instructions and cultured for 48 h.
For GSIS, islets or MIN6 cells were incubated with glucose (2.5 mM) for 4 h prior to incubation with glucose (2.5 mM or 16.7 mM) for 2 h.
For palmitate-stimulated insulin secretion, islets or MIN6 cells were incubated with palmitate (0.5 mM) and glucose (2.5 mM) for 4 h prior to incubation with glucose (2.5 mM or 16.7 mM) for 2 h.
The supernatants were collected and measured by using the Mouse Insulin ELISA Kit (Mercodia) according to the manufacturer’s instructions. The insulin secretion level was normalized to the total cellular protein content. For insulin content, the islets and MIN6 cells were extracted with acid ethanol. The insulin concentration of the extraction was measured by using the Mouse Insulin ELISA Kit and normalized by the total cellular protein content. The protein contents of mouse islets or MIN6 cells were quantified by using a BCA kit (Vazyme).

Echinococcus multilocularis were cultured in DMEM medium with 1% penicillin-streptomycin (without FBS) in an incubator containing 5% CO₂ at 37 ℃. After 8 h, the culture medium was removed, and fresh culture medium (without FBS) was added. ESPs of protoscoleces were obtained at 24 h. Then, the protein concentration was determined by BCA kit (Vazyme, China). The endotoxin in ESPs was removed using Pierce High-Capacity Endotoxin Removal Resin (Thermo, USA), and the amount of endotoxin was determined by the ToxinSensor^™ Chromogenic LAL Endotoxin Assay Kit (GenScript, USA). The amount of endotoxin concentration was < 0.05 EU/ml.

The total protein was extracted in ice-cold RIPA lysis buffer (Solarbio, China) containing PMSF (Solarbio, China) and Phosphatase Inhibitor Cocktail (Epizyme, China) and then sonicated and centrifuged (12,000 ×g for 15 min, 4°C) to collect supernatants. Protein concentration was detected with a BCA kit (Vazyme, China). Lysates of cells or pancreatic tissue were separated by electrophoresis at 10 or 12.5% SDS-polyacrylamide gel and transferred to Immobilon-P membrane (Millipore, USA) which was blocked with 5% BSA for 1 h at room temperature. Primary antibodies applied were as follows: PSGL-1 (NB100-78039, 1:500, NOVUS Biologicals, USA), p-Syk (AF8404, 1:2000, Affinity, USA), PAD4 (ab214810, 1:1000, Abcam, USA), Histone H3 (citrulline R2+R8+R17, ab5103, 1:1000, Abcam, USA), GAPDH (as internal control, 10494-1-AP, 1:5000, Proteintech, China) and β-actin (as internal control, 20536-1-AP, 1:5000, Proteintech, China). ECL detection system was used to visualized the protein bands on the membrane and the grayscale analysis of bands was calculated using ImageJ.