Anti p57kip2

Tissue (1 × 2 mm) or cell line lysates were manually homogenized (PYREX homogenizer; VWR, Radnor, PA, USA) and prepared as previously described [32 (link)]. Supernatant protein was quantified (Pierce BCA protein assay kit; Thermo Scientific, Rockford, IL, USA), total protein lysates (15 μg) were denaturated in SDS sample buffer, separated on an SDS-PAGE gel [10% or 4%–20% (mTOR and 4E-BP1)], and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA; pore size 0.45 mm). Primary antibodies included anti-NAP1L1 (Abcam, Cambridge, MA, USA), anti-p57^Kip2, anti-menin-1, anti-phospho-ERK, anti-ERK, anti-phospho-mTOR, anti-mTOR, anti-phospho-S6K1, anti-S6K1, anti-phospho-4E-BP1, and anti-4E-BP1 (all from Cell Signaling Technology, Beverly, MA, USA). Immunodetection was performed using Western Lightning™ Plus-ECL (PerkinElmer, Waltham, MA, USA). Protein levels were confirmed with β-actin (Sigma-Aldrich, St. Louis, MO, USA). The optical density of the appropriately sized bands was measured using ImageJ software (NIH, Bethesda, MD, USA). The ratio between target protein expression and control protein was calculated.

Antibodies used in this study were commercially obtained as the following: anti‐PAI-1 (C-9) antibody from Santa Cruz Biotech; anti‐p21 Waf1/Cip1 (2947 S), anti-p15 INK4B (4822 S), anti-p57/Kip2 (2557 S), anti-p-Smad2 (Ser465/467) /Smad3 (Ser423/425) (8828 S), anti-Smad2/3 (8685 S), anti-Smad4 (38454 S), anti‐HA (3724), anti-Myc (2276 S), anti-Caspase-3 (2662 S), anti-PARP (9532 S), anti-p-Tyrosine/P-Tyr-100 (9411 S), anti-ABL1 (2862 S), anti-Ac-Histone H3 (K9) (9649 S), anti-Ac-Histone H3 (K18) (9675 S) antibodies from Cell Signaling Technology; anti-KAT3B/p300 (3G230), anti-Histone H3 (ab1791) antibodies from Abcam; anti-GAPDH (G8795), anti-β-actin (A5441), anti-FLAG (F3165), rabbit IgG (I5006) and mouse IgG (I5381) from Sigma‐Aldrich. Polyclonal antibodies against Smad4 pY195, pY301, or pY322 were generated by GL Biochem (Shanghai) Ltd. TGF-β (TGFB1-100) was purchased from StemRD, SB431542 (S4317) from Sigma‐Aldrich and Gleevec/imatinib (CDS022173) from Sigma‐Aldrich, respectively.