Fusion solo

On day 8 of cell differentiation, whole cell protein lysates from differentiated cells were prepared, resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Target proteins, including phospho-protein kinase B (P-Akt), Akt, phospho-extracellular signal-regulated kinase (P-ERK), ERK, phospho-c-Jun N-terminal kinase (P-JNK), JNK, phospho-P38 (P-P38), P38, peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT/enhancer-binding protein alpha (C/EBP-α), C/EBP-β, glucocorticoid receptor (GR), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were detected using primary antibodies and horseradish peroxidase-labeled anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Target proteins were visualized using ECL Plus Western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA). Protein levels were determined densitometrically using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany), as previously described [53 ].

INS-1 cells and C2C12 cells were plated in 6-well plates and cultured overnight, then treated with schisandrin C for 24 h. Subsequently, the cells were lysed with RIPA buffer (Cell Signaling, Danvers, MA, USA) for 20 min. Equal amounts of protein were separated by size using 10% sodium dodecyl sulfate-polyacrylamide gel [38 (link)]. Separated proteins were transferred by electroblotting to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed with the primary antibodies (Cell Signaling) overnight at 4 °C, then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (Cell Signaling) for 1 h at 4 °C, and with enhanced chemiluminescence reagent (GE Healthcare UK Limited, Buckinghamshire, UK) for 5 min at room temperature. Proteins were detected using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany).

The expression of proteins was measured by Western blot analysis. After treatment with compounds 2, 3, and 9 for 24 h, an equal amount of protein lysate (20 μg per lane) from INS-1 cells was added. After 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were probed with primary antibodies against PPARγ, P-IRS-2 (Ser731), IRS-2, P-PI3K, PI3K, P-Akt (Ser473), Akt, PDX-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Danvers, MA, USA) for 1 h at 4 °C, followed by horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (Cell Signaling, Boston, MA, USA) for 1 h at 4 °C. The probed blots after treatment with an enhanced chemiluminescence reagent (GE Healthcare UK Limited, Buckinghamshire, UK) were visualized using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Proteins were extracted using radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH 7.4; 0.5% (v/v) NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄ and cOmplete and Mini, EDTA-free protease inhibitor cocktail, the latter used as instructed by the manufacturer (Roche, Mannheim, Germany)). Concentrations were determined using a Bradford assay according to the manufacturer's instructions (BioRad, Hercules, CA, USA). They were then analysed by SDS-PAGE/ qWB. Membranes were blocked using 5% (w/v) non-fat milk/TBS-Tween 20, which was the buffer also used to dilute secondary antibodies. Primary antibodies were diluted in 5% (w/v) BSA/ TBS-Tween 20. WBs were analysed using the quantitative luminescence system FUSION Solo (PEQLAB, Sarisbury Green, UK). Images were analysed using Image Studio Lite software (LI-COR Biosciences, Lincoln, NE, USA) (www.licor.com/islite). The WB images have been cropped for clarity purposes: that has in no way altered the essence of the data obtained in three independent experiments.

For Western blot analysis, INS-1 cells (4.75 × 10 ⁵ cells/well) were seeded in 6-well plates and incubated for 24 h, followed by treatment with the compounds for 24 h. Total protein was extracted by the addition of RIPA buffer (Cell Signaling, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride for 20 min at 4 °C. This was followed by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto polyvinylidene difluoride membranes by electroplating. The membrane was probed with the primary antibodies against peroxisome proliferator-activated receptor-γ (PPARγ), insulin receptor substrate-2 (IRS-2) (Ser731), IRS-2, pancreatic and duodenal homeobox-1 (PDX-1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), followed by horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody. ECL Plus Western blotting detection reagents (GE Healthcare, Little Chalfont, UK) were used to visualize the probed membranes, which were analyzed using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany).

ME-CSCs and ACSCs were stimulated as described above followed by harvesting via trypsination and subsequent lysis with lysis buffer (0.01 M Tris, 3 mM EDTA, 1% SDS) for 10 min at RT and denaturation for 10 min at 95 °C. The Protein quantification was performed using of Roti-Quant (Carl Roth, Karlsruhe, Germany) following manufacturer’s guidelines. Samples were mixed with 4× loading dye, incubated for 5 min at 95 °C and subjected to electrophoresis on 10% denaturing SDS polyacrylamide gels and subsequently transferred to a nitrocellulose membrane using the Biometra Fastblot B34 blotter (Analytik Jena AG, Jena, Germany). Blocking was subsequently performed with 10% milk powder and 0.1% Tween-20 in PBS for 30 min followed by application of the first antibody against A20 (A-12, sc-166692, Santa Cruz Biotechnologies) and overnight incubation at 4 °C. Afterwards, HRP-linked secondary antibody (515-035-003, Dianova, Hamburg, Germany) was applied for 1.5 h at RT. Visualization was performed via enhanced chemiluminescence using Fusion Solo (PEQLAB, Erlangen, Germany), while GAPDH (sc-32233, Santa Cruz Biotechnologies) served as loading control.