C myc sc 764

Cells were harvested and washed twice with PBS. Cell lysis was performed on ice for 25 min, in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, pH 8.0) containing a protease inhibitory cocktail (Roche). Insoluble material was pelleted by centrifugation at 16,000× g at 4°C for 3 min. Protein concentrations were determined using the Bradford assay (Bio-Rad). Thirty micrograms extract was mixed with 4× Laemmli buffer (40% glycerol, 240 mM Tris/HCl, pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% β-mercaptoethanol), denatured at 96ºC for 5 minutes, separated by SDS-PAGE, and transferred to nitrocellulose membranes (PROTRAN-Whatman, Schleicher&Schuell). The membranes were blocked with 5% non-fat dry milk in TBS-T for 60 min, incubated with primary antibodies overnight at 4°C, washed three times with TBS-T for 10 min, incubated with the peroxidase-conjugated secondary antibody (1:2000; Amersham Biosciences) in TBS-T with 5% non-fat dry milk for 60 min, and washed three times with TBST for 10 min. Immunoreactive proteins were detected using Supersignal West Dura HRP Detection kits (Pierce). The primary antibodies used were: p16^Ink4a (Santa Cruz); p19^Arf (Ab80 Abcam); p15^Ink4b (Santa Cruz); β-catenin (clone 14, BD Biosciences); p53 (sc-6243 Santa Cruz); p21 (BD Pharmigen); c-Myc (sc-764 Santa Cruz); β-actin (ab8226, abcam).

The miR‐6125 precursor overexpression plasmid (HmiR1561‐MR03) and its control plasmid were purchased from Genecopoeia (Guangzhou Science Park, Guangzhou, China). The miR‐6125 micrOFFTM inhibitor (S1127) and its control reagent (U0709) were purchased from RIBOBIO Company (Guangzhou, China). YTHDF2, β‐catenin overexpression plasmid, shGSK3β plasmid and control plasmids were constructed by Miaoling Company (Wuhan, China), and the GFP‐Cyclin D1 plasmid was constructed in the laboratory. Cycloheximide was obtained from Calbiochem (San Diego, CA, USA). Antibodies against Cyclin D1 (2968), c‐Jun (9165), p‐c‐Jun (Ser63) (2361), p‐c‐Jun (Ser73) (3270), SP1 (9389), HA (3724), p‐β‐catenin (Ser33/37Thr41) (9561), GSK3β (12456), Flag (14793) and β‐catenin (8480) were purchased from CST (Boston, MA, USA). Antibodies against GFP (SC‐9996), CDK4 (SC‐260), p21 (SC‐397), p27 (SC‐1641), CDK6 (SC‐7961) and c‐Myc (SC‐764) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against GAPDH (Ab0037) and β‐Actin (Ab0011) were purchased from Abways Technology (Shanghai, China). Antibodies against YTHDF2 (24744‐1‐AP) were purchased from Proteintech (Wuhan, China).

Antibodies used in this study included IKKα (IMG-136A) from IMAGENEX; Lamin B (sc-6216), cytokeratin 5 (K5, sc-17090), p50 (sc-1109), c-myc (sc-764), and IgG (sc-2025) from Santa Cruz Biotechnology; p65 (8242), c-Rel (12707), and E-cadherin (24E10) from Cell Signaling Technology; Sox2 (245610) from R&D Systems; Twist1 (T-6451) and β-actin (A-5441) from Sigma-Aldrich; TNFR1 (ab111119 and ab19139) and histone H3 (acetyl K27, ab4729) from Abcam; UBCH10 (NBP2-20782) from Novus Biologicals; K5 (PRB-160P) from Babco; α-Tublin (ab4074) and histone H3 (acetyl K27, ab4729) from Abcam.