Anti dicer

HQ17(3)-treated cells in 6 cm dishes were rinsed twice with phosphate buffered saline (PBS). Two hundred microliters of RIPA lysis buffer (50 mM tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 2 μL of 100x protease inhibitor complex (Calbiochem Nottingham, UK) was added. The cells were scraped from the Petri dish and transferred to an Eppendorf tube. The tube was placed on ice for 30 min with constant vortex. Cell lysate was centrifuged at 15,000 ×g for 30 min. Sixty micrograms of protein from the supernatant was loaded on 8% or 12% SDS-polyacrylamide gel, followed by western blot analysis. Anti-pRB antibody (Cat. number 9309P, Cell Signaling, Danvers, MA, USA), anti-Dicer (Cat. number ab14601, Abcam, Cambridge, UK), anti-PTEN (Cat. number 9552, Cell Signaling), c-Myc (Cat. number SC-40, Santa Cruz Biotechnology, Inc., Dallas, Texas), antibody against β-actin (Cat. number ACTB12-M, Alpha Diagnostic International, Inc., San Antonio, Texas, USA), and anti-mouse IgG secondary antibody conjugated with peroxidase (A9044, Sigma) were used in the western blot analysis. The immunoreactive bands were revealed by ECL system (Perkin Elmer, Inc., Boston, MA, USA) and developed on X-ray films.

Proteins were extracted by harvesting cells in lysis buffer (50 mM Tris–HCl, 137 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× protease inhibitor cocktail mix without ethylenediaminetetraacetic acid (EDTA), pH 8.0) [14 (link)] and incubating the lysates for 15 min on ice. The lysates were clarified by centrifugation at 16,000× g for 15 min at 4 °C. Protein concentration of the cleared lysates was determined by the method of Bradford using the Bio-Rad (Hercules, CA, USA) dye reagent, with bovine serum albumin as standard. Protein extracts were analyzed by Western blot using mouse monoclonal anti-Dicer (Abcam) [16 (link)] and rabbit polyclonal anti-Flag M2 (Sigma, St. Louis, MO, USA) primary antibodies.

The total cell lysate was subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore). Blots were incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies and detection with ECL plus reagents (GE Healthcare, Chicago, IL). Primary antibodies used were anti-Dicer (ab14601; Abcam, Cambridge, UK), anti-γ-Tubulin (BM1606; BosterBio, Wuhan, China), and anti-GAPDH (2188; Cell Signaling Technology, Danvers, MA). Relative Dicer expression levels were calculated via densitometry and normalized to GAPDH expression using Image J software (http://rsb.info.nih.gov/ij/).

Cells were lysed in Laemmli buffer supplemented with protease inhibitors (complete tablet; Roche) and phosphatase inhibitors (PhosSTOP tablet; Roche) and analyzed using Western blotting with the following primary antibodies: anti-DICER (Abcam); anti-AGO2 (Abcam); anti-α-Tubulin (Abcam), mouse monoclonal to phosphor-Histone H2AX (Ser 139) clone JBW301 (Thermo Fisher Scientific), rabbit polyclonal to Histone H2A (Thermo Fisher Scientific), rabbit polyclonal to phosphor-Chk2 (Thr68) (Cell Signaling, Denvers, MA, USA), mouse monoclonal to Chk2, clone 7 (Thermo Fisher Scientific), rabbit polyclonal to phosphor-Chk1 (Ser 354) (Cell Signaling) and mouse monoclonal to Chk1 (Santa Cruz, Heidelberg, Germany). Primary antibodies were revealed with peroxidase-conjugated goat anti-mouse (Jackson ImmunoResearch Laboratories) and an enhanced chemiluminescence system (Super Signal West Pico Pierce or Super Signal West Dura Extended; Thermo Fisher Scientific).