M per reagent

Protein extraction was performed using M-PER reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions to lyse the cells, and protein concentrations were measured with DC Protein Assay (Bio-Rad); 15–20 µg of a protein sample was loaded into the precast 10% Mini-PROTEAN® TGX Stain-Free^TM Gels (Bio-Rad). After the electrophoresis run, Trans-Blot® Turbo^TM Pack (Bio-Rad) was used to transfer the proteins from the gel to the nitrocellulose membrane. Transfer was done with the Trans-Blot® Turbo^TM Transfer system according to the manufacturer’s instructions (Bio-Rad). A prestained protein ladder, PageRuler Plus (#26619, Thermo Fischer Scientific), was used as a protein size marker. We utilized antibodies against SOX11 (1:1,000) (HPA000536, Sigma-Aldrich, Lot # BB107024) and Histone H3 (1:75,000) (ab4729, Abcam, Cambridge, UK, Lot # GR167613-1). Horseradish peroxidase conjugated anti-rabbit (1:2,000) (P0217, Lot # 00069121) was used as a secondary antibody (Agilent Technologies, Santa Clara, CA, USA). Chemiluminescence reaction by Amersham ECL reagent was detected with ChemiDoc^TM XRS+using Image Lab^TM Software (Bio-Rad).

Glucose-stimulated insulin secretion (GSIS) assay was performed 48-h post-transfection. Briefly, cells were washed twice with secretion assay buffer (SAB), then normalized for 2 h.
Glucose stimulation was done by incubating them in 1 ml SAB (pH 7.2, 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.16 mM MgSO₄, 20 mM HEPES, 2.5 mM CaCl₂, 25.5 mM NaHCO₃ and 0.2% Bovine Serum Albumin) containing either 2.8 mM glucose plus secretagogue, either 16.7 mM glucose, 35 mM KCL or 10 mM α-ketoisocaproic acid (α-KIC) for 1 h as previously described^{41 (link)}. Secreted Insulin was determined using a rat insulin ELISA kit (Mercodia, Sweden).
For measurements of insulin content, the total protein was extracted from transfected cells using an M-PER reagent (Thermo Fisher Scientific) and quantified by Pierce BCA protein assay kit (Thermo Fisher Scientific). Diluted protein (1:250) was then used to evaluated insulin content using a rat insulin ELISA kit (Mercodia, Sweden). Finally, insulin content was normalized to the total amount of protein.

The deamination assay described by Byeon et al.27 (link) was used. Whole-cell lysates were prepared using M-PER reagent (Thermo Fisher) with 1 × Halt protease and phosphatase inhibitor cocktail (Thermo Fisher). Briefly, 180 nM 5′ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (Integrated DNA Technologies) was incubated at 37 °C for an hour with 10 μl lysate and 10 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM dithiothreitol (DTT) and 1 mM EDTA in a volume of 50 μl. The reaction was stopped by adding 40 μg proteinase K (Life Technologies) and incubating it for 20 min at 65 °C. Ten microlitres of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 10 μl of 1 N HCl, the reaction (10 μl) was electrophoresed on a 10% denaturing polyacrylamide gel. Typhoon 9400 Imager (GE Healthcare) was used to scan the gel in fluorescence mode.

The expression of p65 NF-κB and pro-oxidative enzymes was quantified in astrocytes incubated with DHA and treated with 1L-1β (10 ng/ml) for one hour and 24 h, respectively. Next, the cells were washed with PBS and lysed in M-PER Reagent for whole-cell lysate and in NE-PER Reagents (Thermo Fisher Scientific) for cytoplasmic and nuclear fractions. The protein samples (30 µg) were separated on SDS–PAGE, transferred onto nitrocellulose membranes (Millipore) and assayed with anti-p65 NF-κB, anti-COX-2 and anti-iNOS antibodies (Santa Cruz Biotechnology, Inc.). Immune complexes were detected with a Super Signal West chemiluminescent reagent (Thermo Scientific–Pierce). Anti-β-actin and anti-histone H3 (Santa Cruz Biotechnology, Inc.) antibodies were used as a loading control. The immune-reactive signals were quantified by densitometry using Image-J software and normalized to β-actin, or histone H3. Western blot analysis was performed in duplicate. The total protein concentration was measured by modified Lowry’s method and the concentration was read from a BSA standard curve.

Cells were lysed using M‐PER reagent (Thermo Fisher 78501) with complete protease inhibitor cocktail (Sigma, 04693159001) and clarified by centrifugation. Extracted proteins (20 µg) were separated on a precast 4–12% gradient SDS–PAGE gels (Expedeon, NBT41212), transferred to immobilon PVDF (Millipore, IPFL00010) and probed using antibodies for ATG16L1 (MBL M150‐3), LC3A/B (Cell Signaling 41085) and actin (Sigma, A5441). Primary antibodies were detected using IRDye‐labelled secondary antibodies (LI‐COR biosciences, 926‐32211, 926‐68020) and visualised by Odyssey infrared system (LI‐COR).