Rabbit phospho erk

20 MTs pairs per genotype from female flies were dissected in PBS, placed in 30 μl 2xSDS sample buffer (Thermo Scientific, #39001) containing 5% 2-Mercaptoethanol at 100 °C for 10 minutes, ran 4ul on a 4%-20% polyacrylamide gel (Bio-Rad, #4561096), and transferred to an Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). Membranes were blocked by 5% skim milk in 1x Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) at room temperature for 30 minutes. The following primary antibodies were used: mouse anti-tubulin (Sigma, T5168, 1:10,000), rabbit anti-JNK Antibody (D-2) (Santa Cruz, sc-7345, 1:1000), rabbit phospho-JNK (Cell Signaling, 4668 T, 1:1000), rabbit anti-ERK Antibody (Cell Signaling, 4695, 1:1000), rabbit phospho-ERK (Cell Signaling, 4370, 1:1000). After washing with TBST, signals were detected with enhanced chemiluminescence (ECL) reagents (Amersham, RPN2209; Pierce, #34095). Western blot images were acquired by Bio-Rad ChemiDoc MP.

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).

Peripheral blood mononuclear cells were isolated from healthy donors using a Ficoll–Hypaque gradient. T cells were obtained by negative isolation using the Pan T cell Isolation Kit (Miltenyi Biotech). Cells were taken in RPMI 1640 supplemented with 10% FCS and were rested for 1 h at 37°C in the presence or absence of different concentrations of the inhibitor AX-024 (36 (link)). Cells were left unstimulated or stimulated with 10 µg/ml anti-CD3 antibody UCHT1 for 2 min or 5 min and were fixed with 2% paraformaldehyde for 30 min on ice. Subsequently, cells were permeabilized with 87.7% methanol for 30 min on ice and stained with rabbit anti-phospho-ZAP70 (Cell Signaling) or rabbit phospho-Erk (Cell Signaling) overnight. Next, cells were stained with biotin-labeled anti-CD3 (UCHT1; BioLegend), PE-labeled anti-γ/δTCR antibodies (Life Technologies), and DyLight-labeled anti-rabbit IgG (Thermo Scientific) and subsequently with eFluor 450-labeled Streptavidin (eBioscience). Cells were measured by flow cytometry and gated on CD3- and TCRγδ-positive cells for analysis.