Anti cdk4

Protein levels of the genes were detected through western blot. Briefly, cells were lysed using RIPA buffer for 1 h on ice, the protein concentrations were determined via a BCA assay kit (Boster, Wuhan). The proteins were separated by SDS-PAGE. 50 μg of protein and 4×loading buffer were boiled for 10 min and separated by SDS-PAGE (10% separating gel and 5% stacking gel), the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) that were subjected to blocking by 5% skimmed milk for 1 h at room temperature, then incubated with the specific antibodies at 4 °C overnight. The goat anti-mouse and goat anti-rabbit second antibodies were got from Odyssey. The relative amount of gene product was normalized to GAPDH levels.
Proteins were detected by using anti-FAM84B (Proteintech, 18421-1-AP), anti-NPM1 (Proteintech, 60096-1-Ig), anti-CCND1 (Proteintech, 60186-1-Ig), anti-CDK4 (Proteintech, 11026-1-AP), CDK6 (Proteintech, 14052-1-AP), anti-FLAG (Cell Signaling, #14793), anti-HA (Abcam, 9110), anti-GST (Proteintech, 66001-2-Ig), anti-pRb (Cell Signaling, #8516), anti-CDKN2A (10883-1-AP), anti-E2F1 (12171-1-AP) and anti-GAPDH (Proteintech, 60004-1-Ig).

Western blot assays were conducted as previously described.8 The associated primary immunoblotting antibodies were as follows: anti‐GAPDH, anti–E‐cadherin, anti–N‐cadherin, anti‐Vimentin, anti‐CDK6, anti‐CCND1, anti‐CDK4, anti‐NOP14, anti‐MMP9, anti‐MMP2, anti‐E2F1, anti‐MET, anti‐Parp‐1, (Proteintech Group), anti‐Caspase 3, anti‐DNMT3B (Cell Signaling Technology) and anti‐SP1 (Santa Cruz Biotechnology).

The total protein was extracted from A549 cells using RIPA buffer containing protease inhibitors and separated through Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Then, proteins were transferred to a PVDF membrane. After blocked with 5% fat‐free milk for 1 hour, the sample was incubated with primary antibodies for 1 hour and then with secondary antibodies for 1 hour. The following primary antibodies, namely anti‐Syncytin 1 (1:500), anti‐SP1 (1:1000) anti‐Active‐Caspase3 (1:1000), and anti‐Active‐Caspase9 (1:1000) were purchased from Abcam; anti‐Cyclin D1 (1:1000), anti‐Nusap1 (1:1000), anti‐CDK6 (1:1000), anti‐CDK4 (1:1000), anti‐Bcl2 (1:1000), anti‐Bax (1:1000), anti‐β‐catenin (1:1000), anti‐Vimentin (1:1000), anti‐E‐cadherin (1:1000), anti‐N‐cadherin (1:1000), anti‐P70 (1:1000), and anti‐GAPDH (1:5000) were purchased from Proteintech Group; anti‐p‐AKT (1:1000), anti‐p‐mTOR (1:1000), and anti‐p‐Erk1/2 (1:1000) were purchased from Cell Signaling Technology.

HEK-293T cells were transfected with plasmids carrying Flag tagged CLK1 (Flag-CLK1) cDNA and HA tagged SRSF5 (HA-SRSF5) cDNA. At 24 h after transfection, cells were harvested to assess the protein–protein interaction between CLK1 and SRSF5. For immunoprecipitation and Western blot assay, the nuclear and total cellular protein fractions were extracted with RIPA Lysis Buffer (Beyotime, China), as previously described [28 (link)]. The primary antibodies used in this study included anti-Flag (Cat. No. F7425, Sigma-Aldrich, USA), anti-HA (Cat.No.MMS-101P, Biolegend, USA), anti-CLK1 (Cat.No.sc-515897, Santa Cruz, USA), anti-SRSF5 (Cat. No. ab67175, ab67175, Abcam, USA), E-cadherin (Cat. No. ab231303, Abcam, UK), anti-P21 (Cat. No.10355–1-AP, Proteintech, USA), anti-CDK4 (Cat. No. ab108357, Abcam), and anti-N-cadherin (Cat. No. ab207608, Abcam) antibodies. The anti-phosphorylation –SRSFs was constructed in GenScript Inc (Nanjing, China).

Total proteins were extracted by lysing cells in 6‐well plate with 60 μL RIPA buffer supplemented with protease inhibitors (#G2006, Wuhan Goodbio Technology) and phosphatase inhibitors (#G2007, Wuhan Goodbio Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). One microgram of proteins was separated by 10% SDS‐PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). The membrane was blocked with 5% Non‐Fat Milk (#A600669, Sango Biotech) for 1 hour at room temperature and incubated with specific antibody at 4°C overnight followed by HRP‐conjugated secondary antibody incubation for 1 hour at room temperature. Images of membrane with proteins were taken with imager (Bio‐rad ChemiDoc MP, Bio‐rad). The antibodies are recorded: anti‐GAPDH (#60004‐1‐lg, Proteintech), anti‐YTHDF2 (#24744‐1‐AP, Proteintech), anti‐E‐cadherin (#20874‐1‐AP, Proteintech), anti‐N‐cadherin (#66219‐1‐AP, Proteintech), anti‐VIMENTIN (#10366‐1‐AP, Proteintech), anti‐MMP9 (#10375‐2‐AP, Proteintech), anti‐MMP2 (#10373‐2‐AP, Proteintech), anti‐SNAIL (#13099‐1‐AP, Proteintech), anti‐CDK6 (#14052‐1‐AP, Proteintech), anti‐CCND1 (#26939‐1‐AP, Proteintech), anti‐CDK4 (#11026‐1‐AP, Proteintech), anti‐KLF4 (#12173S, Cell Signaling Technology), anti‐SLUG (#C1967, Cell Signaling Technology), anti‐METTL3 (#ab195352, Abcam) and anti‐SETD7 (#ab14820, Abcam).

Jinfukang oral liquid freeze-dried powder was prepared and detected the fingerprint (Additional file 2: Table S2) by Professor Yu Jin of East China University of Science and Technology (Shanghai, China). A cell counting kit-8 (CCK-8) was obtained from Dojindo. The Annexin V-FITC apoptosis detection kit and propidium iodide (PI) were purchased from BD Pharmingen. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) and the antioxidant NAC were purchased from Sigma. A caspase-3 detection kit was purchased from Biovision. The anti-γ-H2AX, anti-p-ATM, anti-p-ATR, anti-PARP1, goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP antibodies were purchased from Cell Signaling Technology. The anti-p53, anti-p21, anti-CDK4, anti-Cyclin D, anti-Cyclin E, anti-Fas, anti-Survivin, and anti-β-actin antibodies were purchased from Proteintech.

Protease inhibition was used to extract total protein from cell lysis of MKN-45 and MGC-803. Bicinchoninic acid protein assay kit was used to measure protein concentration. Protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in appropriate concentration and transformed onto polyvinylidene fluoride membranes. After blocking for 1 h at 4 ℃ using tris-buffered saline with Tween^® 20 (TBST) brewed skim milk powder, the membrane was incubated overnight with the anti-NEK7 (ab13514, abcam, UK) antibody and anti-GAPDH (ab8245, abcam) antibody, which was diluted to an appropriate concentration. Then, after washing, the membrane was incubated with the second antibody at 4 ℃ for at least 1 h and washed by TBST three times. The anti-CDK4 (Cat No. 11026-1-AP), anti-CCND2 (Cat No. 10934-1-AP), anti-KIF3A (Cat No. 13930-1-AP), anti-AKT3 (Cat No. 21641-1-AP), and anti-PRKG1 (Cat No. 21646-1-AP) antibodies were purchased from the Proteintech Group (IL, USA). Signals were detected using a chemiluminescence system (SensiCapture imaging system, Peiqing Technology Co. LTD, China).