Anti dyrk2

Cells and tissues were lysed by radioimmunoprecipitation (RIPA) buffer without sodium deoxycholate (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40) with various inhibitors (1 mM Na₃VO₄, 1 mM AEBSF, 1 mM DTT, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM NaF, 1 μg/ml pepstatin A) and cOmplete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Protein concentrations in the supernatants were detected by the DC™ protein assay kit (Bio-Rad Laboratories, Hercules, USA). The equal amounts of proteins were separated by SDS-PAGE. Proteins were detected by the following antibodies diluted by CanGetSignal Solution 1 (Takara Bio Inc.); anti-GAPDH (Bethyl Laboratories, Montgomery, TX, USA), anti-DYRK2 (Sigma-Aldrich), anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), anti-Myc, anti-Myc-Ser62 (Abcam), anti-FLAG (Sigma-Aldrich), anti-Hras, anti-cyclin E, anti-cyclin D1, and anti-cyclin D2 (Santa Cruz Biotechnology, Dallas, TX, USA). Membranes were developed by ImmunoStar LD (Fujifilm Wako Pure Chemical Co., Osaka, Tokyo) and imaged using FUSION SOLO 4 M, and analysed using Fusion Capt Advance software (M&S Instruments Inc., Osaka, Tokyo).

Cells were washed twice in chilled PBS and resuspended in lysis buffer (50 mmol/L Tris‐HCl, pH 7.6; 150 mmol/L NaCl; 10 mmol/L NaF; 1 mmol/L Na₃VO₄; 1 mmol/L PMSF; 1 mmol/L DTT; 10 μg/mL aprotinin; 1 μg/mL leupeptin; 1 μg/mL pepstatin A; 1% NP‐40). Cell extracts were centrifuged for 5 min at 4°C. Supernatants were separated by SDS‐PAGE and transferred to nitrocellulose membranes. The membranes were incubated with anti‐CDK14 (Santa Cruz Biotechnology), anti‐DYRK2 (Sigma‐Aldrich), anti‐AR (Santa Cruz Biotechnology), anti‐TUBULIN (Sigma‐Aldrich) or anti‐GAPDH (Santa Cruz Biotechnology) antibodies at a 1:1500 dilution. Immune complexes were incubated with secondary antibodies and visualized using ImmunoStar (Wako, Osaka, Japan) or Western Lightning Plus‐ECL (PerkinElmer, Norwalk, CT, USA).

Immunoblot analyses were carried out as previously described.
¹⁸ (link) The membranes were incubated with antibodies against anti‐DYRK2 (Sigma‐Aldrich), anti‐p53 (Santa Cruz Biotechnology), anti‐phospho‐p53‐Ser46 (Bio Academia and Cell Signaling Technology), anti‐cleaved poly (ADP‐ribose) polymerase (PARP) (Cell Signaling Technology), anti‐cleaved caspase‐3 (Cell Signaling Technology) and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology). Then membranes were incubated with peroxidase‐conjugated anti‐rabbit IgG (Cell Signaling Technology) or anti‐mouse IgG (Cell Signaling Technology). The dilution ratios were all 1:1000.