Protein extraction kit

Cytoplasmic proteins, cytosolic proteins and total proteins were extracted using a protein extraction kit (abcam, UK) and proteins in all samples were quantified with Bicinchoninic acid protein assay. Proteins of equal amounts from all samples were separated with SDS/PAGE gel (Bio-Rad, USA) and transferred onto PVDF membrane (Bio-Rad, USA). Bands were sealed with 5% skim milk, incubated in primary antibodies (anti-CD22 and anti-GAPDH, CTS, SUA) at 4 °C overnight, then in secondary antibodies (HRP-goat anti-rabbit IgG, CTS, SUA) at room temperature for 1 h, and then examined and analyzed by using ChemiDoc™ XRS + with Image Lab™ Software (Bio-Rad, USA). Due to the significant difference of molecular weight between the reference protein and the target protein as well as the inconsistent conditions for transferring onto PVDF membrane, the gels were cut before transferring, and the incubation of the antibody as well as exposure of the reference protein and the target protein were also carried out separately.

BMDMs were lysed using a Protein Extraction Kit (Abcam) for protein extraction. 20‐µg proteins were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transfer. Membranes were blocked with 5% non‐fat milk in PBST at 4°C overnight. The next day primary antibodies diluted in 1% non‐fat milk in PBST were added for incubation for 2 h at room temperature. After wash, horseradish peroxidase‐conjugated corresponding secondary antibodies were added. Chemiluminescent substrate (Bio‐Rad) was used to visualize the immunoreactive bands. Primary antibodies used in present study were: anti‐IKBα (C‐21, 1:1000), anti‐ERK (K‐23, 1:5000), anti‐phospho‐ERK (E‐4, 1:3000), anti‐MEK1 (C‐18), anti‐JNK1 (C‐17, 1:1000), anti‐p38 (H‐147, 1:1000), anti‐phospho‐JNK (Thr180/Tyr185, 1:1,000), anti‐phosphop38 (Thr180/Tyr182, 1:1000), and anti‐phospho‐MEK1/2 (Ser217/221). The first six antibodies were purchased from Santa Cruz Biotechnology, while others were from Cell Signaling Technology.

The cytoplasmic and nuclear fractions separated using the protein extraction Kit (BioVision, Palo Alto, USA) according to the manufacturer’s instructions. Whole cell lysates were prepared in a buffer containing protease and phosphatase inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany) at 4 °C. Western blotting analysis was performed as previously described^{20 (link)}. Primary antibodies were used: HMGB1, RAGE, β-actin, GAPDH, Lamin B1, phospho-ERK, ERK, and γH2AX (Cell Signaling Technology, USA, except HMGB1 and RAGE from Abcam, USA).

Immunoblotting and subsequent densitometry were performed with mixed plasma membranes, and intracellular membranes were incubated with antibodies against Mrp2 (ab3373, 1:50, abcam) and Na⁺/K⁺-ATPase (#3010, 1:1000, Cell Signaling). Moreover, the crude membranes were prepared according to the protocol of protein extraction kit (BioVision, USA). Equal amounts of crude membranes (40 μg/lane) were separated on 6%-15% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in 5% skim milk and incubated with antibodies against Bsep (sc-25571, 1:200, Santa Cruz), Ntcp (#6522-1, 1:1000, epitomics), Oatp1 (LS-C113034-50, 1:1000, LifeSpan BioSciences), Mrp3(ab3375, 1:50, abcam), Mdr2 (SAB2100008, 1:1000, sigma) and Na⁺/K⁺-ATPase (#3010, 1:1000, Cell Signaling), respectively. Blots were incubated with horseradish peroxidase conjugated secondary antibodies (Santa Cruz) and developed by ECL detection regents (Amersham). The protein bands were quantified by the average ratios of integral optic density (IOD) following normalization to Na⁺/K⁺-ATPase expression.

Min6 cells were treated with medium, 16.7 mM glucose, and a combination of 16.7 mM glucose with STG (10 μg/mL) and PS1 at 0.2, 1, and 5 μg/mL for 30 min. The cells were incubated with Hoechst 33,342, a nuclear dye, plus CM-H₂DCFDA, a dye for cytosolic ROS, and MitoGreen, a mitochondrial tracker, plus MitoSOX, a dye for mitochondrial ROS, and analyzed using confocal microscopy. To measure the ETC C1 and Nox activity, membrane and mitochondrial fractions of the cells were individually extracted (protein extraction kits, Abcam, UK) and tested for the activity of Nox and ETC C1, respectively, as previously described [2 (link)].

Pancreatic islets isolated from the mice were pre‐treated with 3.3 mM (LG) and 16.7 mM (HG) glucose for 30 min. The islets were then incubated with Hochest 33342 plus CM‐H₂DCFDA or MitoGreen plus MitoSOX for additional 30 min. Alternatively, Min6 cells and those infected with lentiviral particles were grown in RPMI medium in the presence or absence of NAC (1 mM) and GHTT (10 μg/ml) for 30 min. The cells were then incubated with CellROX plus Hochest 33342 or MitoGreen plus Mitosox in the presence of 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. The ROS signal of the islets or cells was recorded using confocal microscopy and quantified using the Zen software. To measure serum ROS (Tsai et al, 2011 (link)), the sera of mouse tail veins were incubated with lucigenin and monitored using Victor 3 (Perkin Elmer). Mouse islets were pre‐treated with 3.3 mM (LG) and 16.7 mM (HG) glucose for 30 min. Membrane and mitochondrial fractions of the islets were prepared with protein extraction kits (Abcam, UK) according to the manufacturer’s protocol. The membrane fractions were incubated with lucigenin and NADPH and tested for Nox activity as published (Minkenberg & Ferber, 1984 (link)). The mitochondrial fractions were measured for the activity of ETC I to IV using MitoCheck CI, CII/III and CIV kits based on the manufacturer’s guidelines (Cayman, MI).