Anxa10

Whole protein was extracted using mammalian protein extraction reagent (M-PER) from the cell lines. The proteins were digested using the Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA, USA) and complete protease inhibitor cocktails (Roche, Lewes, UK) according to the manufacturer’s protocols. The digested proteins were separated on 4–15% gradient sodium dodecyl sulfate (SDS)–polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The following primary antibodies were used: Cul4A (Abcam, Cambridge, MA, USA), ANXA10 (GeneTex), and β-actin (Sigma, St. Louis, MO, USA). After incubation with indicated secondary antibodies, the membranes were washed thoroughly and an enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for antigen-antibody detection. The relative intensities of protein bands were analyzed by densitometry using ImageJ 1.46r software (National Institutes of Health, Bethesda, MD, USA).

Formalin-fixed, paraffin-embedded tissues were cut into 4-μm sections, mounted on slides, deparaffinized with xylene, and dehydrated using a gradient ethanol series. Antigen retrieval with citric acid (pH 6.0) at 97 °C for 30 min, followed by treatment with 3% hydrogen peroxide were performed. The slides were incubated overnight at 4 °C with an anti-Cul4A antibody (Abcam, Cambridge, MA, USA) or ANXA10 (GeneTex, Irvine, CA, USA). The IHC data for the specimens were assessed using the semi-quantitative immunoreactive score (IRS). The IRS was calculated by multiplying the staining intensity (0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) by the percentage of the positively stained cells (0 = 0% of cells stained, 1 = less than 10% of cells stained, 2 = 11–50% of cells stained, 3 = 51–80% of cells stained, and 4 = more than 81% of cells stained) as described in [29 ].