Anti ccnd1

Total protein from cells was extracted using RIPA buffer (Pierce, Rockford, IL, USA). The protein concentration was determined using the Bradford method (Pierce). Equal amounts of protein (40 μg) were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS_PAGE) and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in phosphate-buffered saline and Tween-20 for 1 h, membranes were incubated with anti-RAB14, anti-Akt, anti-p-Akt, anti-CCND1, anti-CDK2, or anti-Bax antibody (1 : 2000 dilution; Abcam, Hong Kong, China) as well as anti-GAPDH antibody (1 : 2000; Abcam) at 4°C overnight followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 5,000 dilution) at 37°C for 1 h. Immunoreactive bands were detected using the ECL Plus Detection kit (Pierce).

72 h after transfection, cell protein lysates were separated in 8% or 10% SDS-PAGE gel, followed by subsequently transferred to polyvinylidene difluoride membrane (PVDF). Western blot analysis was performed with monoclonal anti-p53 (Santa cruz), anti-EGFR (Abcam), anti-AKT2 (Abcam), anti-CCND1 (Abcam), and anti-E-cadherin (Abcam) antibodies. Anti-GAPDH antibody (Santa cruz) was used as an internal control. The membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA). Complexes were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and the expression levels of these proteins were evaluated by Quantity One software.

Cells and tissues were lysed, and the proteins were collected. Next, the protein samples were separated and moved to PVDF membranes. After that, the membranes sealed by non‐fat milk were incubated with primary antibodies. Later, a secondary antibody was added. Anti-CAPG (ab155688), anti-GAPDH (ab16891), anti-β-catenin (ab32572), anti-N-cadherin (ab76011), anti-E-cadherin (ab40772), anti-CCND1 (ab16663), anti Cluadin1 (ab180151), anti-ZO-1(ab307799) purchased from Abcam, anti-Snail(A5243), anti-Slug (A1057), purchased from ABclonal.

The total protein from cells was extracted using RIPA buffer (Pierce, Rockford, IL, USA). The protein concentration was determined using the Bradford method (Pierce). Equal amounts of proteins (40 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in phosphate-buffered saline (PBS)–Tween-20 for 1 h, the membranes were incubated with anti-RAB14, anti-Akt, anti-p-Akt, anti-CCND1, anti-CDK2, or anti-Bax-antibody (1:2000 dilution, Abcam, Hong Kong, China), as well as anti-GAPDH (1:2000, Abcam) antibody at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5000) at 37 °C for 1 h. Immunoreactive bands were detected using the ECL Plus Detection kit (Pierce, Rockford, IL, USA).

Immunoblot analysis for U2OS cells was performed as described (61 (link)) using the following antibodies: anti-BMAL1 (14020, Cell Signaling, Danvers, MA, USA), anti-CLOCK (sc-6927, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PER2 (20359-1-AP, Proteintech, Chicago, IL, USA), CRY1 (A302-614A, Bethyl Laboratories, Montgomery, TX, USA), anti-CRY2 (13997-1-AP, Proteintech), anti–REV-ERBα (13418, Cell Signaling), anti–REV-ERBβ (GTX115322, GeneTex, Irvine, CA, USA), anti-CCNB1 (#4138, Cell Signaling), anti-CCND1 (ab134175, Abcam, Cambridge, MA, USA), anti-CDK4 (12790, Cell Signaling), anti-AKT (4685S, Cell Signaling), anti-phospho AKT Ser⁴⁷³ (pAKT) (4060S, Cell Signaling), anti-p44/42 mitogen-activated protein kinase (MAPK) (Erk1/2) (4695S, Cell Signaling), anti–phospho-p44/42 MAPK (Erk1/2)-Thr²⁰²/Tyr²⁰⁴ (pERK) (4370S, Cell Signaling), anti–caspase-3 (9662S, Cell Signaling), anti-HSF1 (sc-17756, Santa Cruz Biotechnology), anti-HSP70 (4872S, Cell Signaling), anti-HSP90AA1 (8165S, Cell Signaling), anti-HSP90AB (7411S, Cell Signaling), anti-HSP90B1 (Grp94) (2104S, Cell Signaling), and anti-TRAP1 antibody (GTX102017, GeneTex). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (sc25778, Santa Cruz Biotechnology), anti-tubulin (ab18251, Abcam), and anti–β-actin (4967S, Cell Signaling) were used as loading control antibodies.