Quantity one v4

Kinetic parameters of Nei, NEIL1 and NEIL2 cleavage of 20-mer substrates X1/C1 and X2/C2 were obtained using AP sites as the lesions. To create AP sites, uracil-carrying substrates (100 pmol) were treated with 10 U of E. coli Ung (SibEnzyme, Novosibirsk, Russia) for 10 min at 37 °C immediately before use. The reaction mixture (10 μL) contained 20 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, 5–500 nM substrate and Nei (5 nM), NEIL1 (5 nM) or NEIL2 (10 nM). After 5 min at 37 °C, 5 μL of formamide-containing dye (80% formamide, 20 mM EDTA, 0.1% xylene cyanol and 0.1% bromophenol blue) was added, and the mixtures were heated for 2 min at 95 °C. The reaction products were resolved by 20% denaturing PAGE and visualized by phosphorimaging (Typhoon FLA 9500, GE Healthcare). The images were quantified using Quantity One v4.6.8 software (Bio-Rad Laboratories, Hercules, CA, USA). K_M and V_max were calculated by non-linear fitting to the Michaelis–Menten equation using SigmaPlot v11.0 (Systat Software, Frankfurt am Main, Germany).

Freshly isolated murine neutrophils were incubated with vehicle, with LPS (1 μg/ml), or a combination of LPS and the fibrate WY-14643 (WY, 10 μM) for 1, and 5 min. Preparation of proteins and subsequent Western blot analysis were performed as described (Tancevski et al, 2010 (link)). Antibodies against ERK, p-ERK, p38, and p-p38 were from Cell Signaling (Danvers, MA). The chemoluminescent reaction was performed using Super Signal West Dura Reagent (Pierce, Rockford, IL), and blots were visualized by Fluor-S-Imager using Quantity One V4.1 software (Bio-Rad, Hercules, CA).

The densitometric analysis was performed using the Quantity One v4.1 software (Bio-Rad, Hercules, CA) or ImageJ and statistical analyses were performed with PRISM 5 software (Graphpad, USA). M2 densitometric values were normalized to GAPDH and expressed as D^M2/D^GAPDH. Means were statistically analysed using the t-test or ANOVA and differences assessed at p<0.05 (*) or p<0.01 (**). SUMO-1 and acetyllysine densitometric values were expressed as D^Ac/D^SUMO with the WT ratio in the siCtrl condition arbitrarily set as 100%.

Equal amounts of total protein were loaded onto 12% SDS/PAGE at 80 V for 80 min, electro transferred to polyvinylidene difluoride membranes by the wet transfer method, and blocked in 5% BSA at 4 °C overnight. Subsequently, the membranes were incubated with an anti‐β‐actin antibody (Santa Cruz, CA, USA; 1 : 1000) and anti‐nucleotide‐binding oligomerization domain‐like receptors 3 (NLRP3) antibody (Santa Cruz, CA, USA; 1 : 1000) at room temperature for 2 h. After washing with TBST, the membranes were incubated with secondary goat anti‐mouse IgG antibody (Santa Cruz, CA, USA; 1 : 500) or goat anti‐rabbit IgG antibody (Santa Cruz, CA, USA; 1 : 500) at room temperature for 1 h. Equal loading of protein in each lane was verified by reblotting the membrane with an anti‐β‐actin antibody (ZSGB, Beijing, China). Then, proteins were detected by chemiluminescence reagent. Protein band density was quantified using Bio‐Rad quantity one v4.62.

Total protein extracted from cells or tissues was lysed, separated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blocked with 5% skimmed milk. The primary antibodies including c-MYC (1:1000, sc-40, Santa Cruz Biotechnology), DNMT3a (1:2000, NB120-13888, Novus Biologicals), PTEN (1:1000, 9559, Cell Signaling Technology), CD9 (1:1000, ab92726), CD63 (1:1000, ab59479), Calnexin (1:1000, ab22595, Abcam), HSP70 (1:1000, 4872, Cell Signaling Technology), and β-actin (1:1000, sc-47778, Santa Cruz Biotechnology), along with secondary antibody (7074, 1:2000, Cell Signaling Technology) were applied to incubate with the membrane. The intensity of the band was quantified by Image analysis system (Quantity One v4.62, Bio-Rad) [38 ].

Protein obtained from mouse GC was extracted using the Total Protein Extraction Kit according to the manufacturer’s instructions. After total protein quantification by a BCA assay, the samples were stored at −80 °C for subsequent use. The total protein per sample was separated using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membrane was treated with a blocking buffer (5% nonfat dried milk in Tris-buffered saline (TBS) containing 0.1% Tween 20) for 1 h at room temperature and incubated overnight at 4 °C with the following antibodies: anti-XBP1 (1:1000), anti-Cyp19 (1:200), anti-Cyp11a1 (1:200), anti-StAR (1:200), anti-Runx2 (1:200), anti-caspase-3 (1:200), anti-cleaved caspase-3 (1:1000), anti-CHOP (1:1000), anti-Bcl-2 (1:200), and anti-β-actin (1:1000). The membranes were washed three times with TBST and then incubated with biotinylated anti-rabbit IgG antibody or biotinylated anti-mouse IgG antibody (1:2000) for one hour at room temperature. Finally, the immunoreactive bands were imaged using a digital microscope (Tanon-4100, Tanon Science & Technology Co., Ltd., Shanghai, China) and densitometric analyses were performed using the Quantity one v4.62 (Bio-Rad, Hercules, CA, USA).