Odyssey system

Protein from 6-month-old E-P72 and E-R72 mammary glands was extracted from using boiling 2x Laemmli sample buffer, and concentrations were determined with the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Equal amounts of total protein were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were probed with following primary antibodies: from Cell Signaling Technologies (CST): CCL2 (1:1000, #2029) GAPDH (1:5000, #2118), Phospho-RB (1:1000, #8516), Phospho-p65 (Ser536) (1:1000, #3033), TNFα (1:1000, #11948), from Santa Cruz Technologies: p53 (1:500, sc-6243) and p21 (1:500, sc-397).
All blots (except GAPDH) were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:2500, CST, #7074) and developed using ECL Prime reagents (GE Healthcare, IL). Images were captured using a FluorChem M imager and quantified with AlphaView software (ProteinSimple, CA). GAPDH blots were incubated with iRDye800CW secondary antibody (1:5000, LI-COR Biosciences 925–3211) and developed and imaged using the Odyssey Li-COR system (LI-COR Biotechnology, NE).

HCT116 cells grown in 24-well plates were pelleted by centrifugation and resuspended in ∼50 μl of 2× protein loading dye (75 mM Tris–HCl, pH 6.8, 1.25 mM EDTA, 20% glycerol, 2.5% SDS, 0.125% bromophenol blue, and 50 mM DTT). 10 μl of each sample was loaded on a 1.5 mm denaturing SDS 13% polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Protran; GE Healthcare) and probed with monoclonal primary antibodies recognising EXOSC8, EXOSC9, p53, karyopherin β1, or GAPDH, diluted in PBS-Triton X-100 (0.1%, vol/vol) containing 2% non-fat dried milk (wt/vol) (Marvel). An IRDye-labelled secondary antibody (LI-COR) was used, and membranes were visualised using the Odyssey LI-COR system (LI-COR). Quantification was performed using ImageQuant software (GE Healthcare) and quantified protein levels were normalised to levels of the loading control, karyopherin. The following antibodies were used: α-EXOSC8 (mouse, (H-8) sc- 393027; Santa Cruz), dilution 1:1,000; α-EXOSC9 (mouse, (D-6) sc-271815; Santa Cruz), dilution 1:1,000; α-p53 (mouse, (DO-1) sc-126; Santa Cruz), dilution 1:500; α-karyopherin β1 (mouse, (H-7) sc-137016; Santa Cruz), dilution 1:2,000; α-GAPDH (mouse, (0411) sc-47724; Santa Cruz), dilution 1:10,000; and secondary antibody Donkey α-Mouse 800CW LI-COR (926-32212), dilution 1:10,000.

Attached cells were collected by gentle scraping for protein extraction. Whole cell lysates were prepared by boiling cells for 10 min in a 2% SDS buffer (2% SDS, 50 mM Tris-HCl pH 6.8, 10% glycerol) supplemented with protease inhibitors (Sigma-Aldrich). Proteins were resolved on 8–10% SDS-PAGE followed by transfer to 0.2 μm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). Western blotting was performed by incubating with appropriate primary antibodies followed by secondary antibodies conjugated to far-red fluorescent dyes (IRDye-680 and -800) and detection using the Odyssey LI-COR system (LI-COR Biotechnology, Lincoln, NE, USA). Blot quantifications were performed using LI-COR software. All data were acquired from at least 3 independent experiments.

Cells were washed once with PBS, lysed in 2 x SDS Laemmli buffer and boiled for 10 min. Proteins in cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes. Membranes were washed once with Tris buffered saline (TBS), blocked in a buffer containing 3% BSA or 5% milk in TBS for 30 min at room temperature and probed with the respective primary and secondary antibodies in blocking solution supplemented with 0.02% SDS and 0.1% Tween 20. Blots were visualized and analyzed using the Odyssey LI-COR system and the LI-COR Odyssey application software. Alternatively, blots were visualized by enhanced chemiluminescence (ECL).

Cell lysates from the NK92 human NK cell line was used as a source of proteins to measure level human 12-LO levels using the Odyssey LI-COR system (Lincoln, NE, USA) as previously described (61 (link)). Nuclear and cytosolic proteins were fractionated and tested separately. Duplicate samples were stained with antibodies recognizing tubulin, ALOX12S, and ALOX15-1.

Total RNA was extracted from mandibular tissues of Irf6 null and wild type murine embryos at E10.5, E12.5 and E14.5 to measure expression level of several genes. RTqPCR experiments were performed as previously described^{55 (link)}. Primers for Twist1, Runx2, Grhl3 and Gapdh were described previously^{56 (link), 57 (link)}. Gapdh expression level was used in each plate for normalization purposes. Four technical replicates of each treatment were used to calculate the average of relative gene expression and the bar represents the standard deviation. Immunoblot was performed in mandibular tissues for the wild type and mutant embryos to detect the total protein levels at E10.5 and E12.5 time points as previously described^{29 (link)}. Odyssey Li-Cor system was used to visualize the protein bands and GAPDH was used as a loading control. We performed multi-variants ANOVA test for the normalized expression data to determine the significant difference among the mean values. A p-value less than 0.05 is considered statistically significant.