C1053

Total protein was extracted from the hippocampus in RIPA lysis (C1053, APPLYGEN, China) buffer containing a protease inhibitor and supplemented with phosphatase inhibitors (11836170001, Roche, Germany), and centrifuged at 12,000 × g for 5 min at 4°C. The protein was quantified by the BCA protein assay kit. Protein lysates were cleared of insoluble material through centrifugation. Proteins were wet transferred to 0.2 μm pore size, hydrophobic PVDF transfer membranes (ISEQ00010, Millipore, Germany), which were blocked using 5% non-fat milk in 1% TBST buffer for 1 h at room temperature. The membranes were incubated overnight using the following primary antibodies: O-GlcNAc (CTD110.6, 1:1000 dilution) Mouse mAb (12938S, CST, USA), GAPDH (1:5000 dilution) Mouse McAb (60004-1, Proteintech, China). All primary antibodies were used in 5% BSA (CZ1006, czkwbio, China). Membranes were washed in 1%TBST (T1082, Solarbio, China) and incubated with the following appropriate secondary antibodies: goat anti-mouse HRP. The secondary antibodies were used at a 1:5,000 and 1:10000 dilution in 5% BSA. Protein bands were visualized following exposure of the membranes to ECL (RM0021, ABclonal, China) and quantified by densitometry analysis using Image software.

VSMCs were washed with PBS, lysed with RIPA lysis buffer containing phosphatase inhibitor (C1053, Applygen Technology, China) for total protein extraction on ice. Following the routine procedures of Western blot, 20 μg total protein per sample was electrophoresed on SDS-PAGE gel, then transferred to PVDF membrane (Invitrogen, USA), and blocked with 5% defatted milk powder at room temperature. The membranes were incubated overnight with the primary antibodies of OPN (1 : 1000, AF0227), SM-MHC (1 : 1000, DF8344) PIK3C2A (1 : 1000, DF2623), LC3 (1 : 1000, AF5402), Beclin-1 (1 : 1000, AF5128), p62 (1 : 1000, AF5384), C/EBPα (1 : 1000, AF6333), MMP-2 (1 : 1000, AF5330), MMP-9 (1 : 1000, AF5228), or GAPDH (1 : 1000, AF7021), followed by the incubation at room temperature with the secondary antibody (1 : 2000). All antibodies were purchased from Affinity Biosciences (Australia). Then, the bands were exposed with ECL solution and visualized by Chemi Doc MP. The bands were analyzed by the ImageJ software with GAPDH as the internal reference.

Western blotting was conducted as previously reported 22 (link). Briefly, we consistently collected PDAC surgical resection pathological specimens without any bias or pre-screening during 2019, from which 42 paired PDAC tissues and paracancer tissues were randomly selected for western blot analysis. 100 mg samples were lysed in radioimmunoprecipitation assay (RIPA) buffer (APPLYGEN, C1053+) containing 1% protease inhibitor and 1% phosphatase inhibitors for 30min on ice. Then, centrifugation at 12,000 × g for 15 min at 4 °C, the supernatant proteins were quantified using BCA assay (Thermo Scientific, BCA protein assay kit, 23225). Thirty micrograms of protein were mixed with 5 × loading buffer and denatured in a 95 °C water bath. Primary antibodies against TIGIT rabbit mAb (Abcam, ab243903, 1:1000), CD155 rabbit mAb (Cell Signaling Technology, #81254, 1:1000), and anti-β-Actin (D6A8) rabbit mAb (Cell Signaling Technology, #8457, 1:1000) were incubated with PDAC tissues overnight at 4°C for the following blot detection.

The islets of KKA^y mice were incubated in RPMI-1640 medium containing vehicle or 100 μg/ml SZ-A for 24 h. MIN6 cells were fasted for 1 h in Krebs buffer (2.8 mM glucose) with or without different concentrations of SZ-A, FAG, or DAB. Then, they were cultured for another 1 h in new Krebs buffer containing 2.8 mM or 16.8 mM glucose combined with SZ-A, FAG, DAB or vehicle. High glucose- and palmitic acid-treated MIN6 cells were coincubated with different concentrations of SZ-A, DNJ or vehicle for 24 h.
Islets or MIN6 cells were homogenized in RIPA lysis buffer (C1053; APPLYGEN, China) containing protease inhibitors (P1265; APPLYGEN, China) and phosphatase inhibitors (P1260; APPLYGEN, China). Protein (10 μg) from each sample was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes for immunoblotting. Rabbit anti-phospho-AMPKα (1:1000, 2535s), rabbit anti-AMPKα (1:1000, 2532s), rabbit-phospho-Erk (1:1000, 9101s), rabbit anti-Erk (1:1000, 9102s), and rabbit anti-HSP90 (1:1000, 4877s) were purchased from Cell Signaling Technology (USA). Goat anti-rabbit IgG/HRP (1:5000, ZDR-5306) was purchased from ZSGB-BIO (China).