Anti c myc

Cells were collected and lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), protease inhibitors). Equal amounts of proteins (40 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis gels and then transferred to a nitrocellulose blotting membrane (PALL Corporation, Mexico). After blocking with 5% non-fat milk, the membrane was incubated overnight at 4 °C with the respective primary antibody. The following antibodies were used: anti-c-MYC (1:1000, 13987S), anti-SLC3A2 (1:1000, 13180S), anti-E-cadherin (1:1000, 3195S), and anti-SLC7A11 (1:500, 12691S) from Cell Signaling Technology (Danvers, MA); anti-β-actin (1:10,000, sc-47778), anti-MTDH (1:250, 517220), and anti-GPx4 (1:500, sc-166570) from Santa Cruz Biotechnology; anti-vimentin (1:1000,v6389) from Sigma-Aldrich; anti-ZEB1 (1:1000, NBP1-88845) from Novus Biologicals USA. Membranes were further incubated with secondary antibody (1:10,000, 7074S or 7076S, Cell Signaling Technology) at room temperature for 2 h. Signal bands were detected using the Bio-Rad ChemiDoc system and densitometry were analyzed with Bio-Rad Image Lab Software (Bio-Rad Laboratories).

Western blot analysis was conducted as previously described [24 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, and anti-c-Met (Epitomics, Burlingame, CA, USA), anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin, anti-c-myc, anti-EGFR, anti-p-EGFR (Tyr1068), anti-GSK3β, anti-p-GSK3β (Ser9), anti-AKT (pan), anti-p-AKT (Ser473), anti-stat3, anti-p-stat3 (Tyr705) and anti-CREB1 (Cell Signaling Technology, Beverly, MA).

Whole cell lysates were prepared in RIPA buffer and loaded onto SDS-PAGE. Western blotting was performed using following antibodies: anti-MITF C5 (Millipore, MAB3747), anti-human GTF2H1 (AbD, MCA4041Z), anti-c-Myc (Cell Signaling, #5605), anti-human CDK7 (Cell Signaling, #2916; Santa Cruz, sc-365075), anti-human phospho-CDK7 T170 (Abcam, ab155976), anti-human MAT-1 (Santa Cruz, sc-6234), anti-human cyclin H (Abcam, ab92376), anti-human CDK2 (Santa Cruz, sc-53220), anti-human RNA polymerase II (Bethyl, A300-653A), anti-human phospho-RNA polymerase II (S2) (Bethyl, A300-654A), anti-human phospho-RNA polymerase II (S5) (Bethyl, A300-655A), anti-human p27 (Santa Cruz, sc-528), anti-HA-tag HRP conjugate (Cell Signaling, #2999), anti-FLAG (Sigma Aldrich, F1804), and anti-β-Actin (Sigma Aldrich, A2228). Anti-mouse immunoglobulins/HRP (Dako, P0447) and anti-rabbit immunoglobulins/HRP (Dako, P0448) were used as secondary antibodies. Proteins were visualized with Amersham ECL solution (GE Healthcare, RPN2106).

Gefitinib (AstraZeneca), Phenylarsine Oxide (PAO) (Sigma‐Aldrich), Filipin III (Sigma‐Aldrich), anti‐EGFR (sc‐373746), anti‐EGFR (4267, Cell signaling), anti‐p‐EGFR (2234, Cell signaling), anti‐STAT3 (sc‐8019), anti‐p‐STAT3 (sc‐8059), anti‐ERK (9102, Cell signaling), anti‐p‐ERK (9101, Cell signaling), anti‐EEA1 (610457, BD Biosciences), anti‐PARP (9542, Cell signaling), anti‐c‐Myc (9402, Cell signaling), anti‐β‐actin (A5316), Goat anti‐mouse IgG (H + L)‐HRP conjugate (1706516, Bio‐Rad), Goat anti‐rabbit IgG polyclonal HRP conjugated (ADI‐SAB‐300, Enzo), Alexa Fluor 488 goat anti‐rabbit antibody (A32731, Thermo Fisher Scientific), and Alexa Fluor 594 goat anti‐mouse antibody (A32742, Thermo Fisher Scientific).

RNA isolation and RT–qPCR analysis were carried out according to previous studies (20 (link)). β-actin served as an internal control. The sequences of the primers used in the experiment are as follows. Human IGF2BP3: 5′- TCGAGGCGCTTTCAGGTAAA-3′ (forward), 5′- AAACTATCCAGCACCTCCCAC-3′ (reverse). Mouse Igf2bp3: 5′- CCTGGTGAAGACGGGCTAC-3′ (forward), 5′- TCAACTTCCATCGGTTTCCCA-3′ (reverse).
Protein extraction and Western blot analysis were carried out according to previous studies (20 (link)). The primary antibodies included rabbit anti-IGF2BP3 (1:1000, Proteintech, Chicago, USA), anti-CCNB1 (1:1000, Shanghai, China) and anti-C-Myc (1:2,000, Cell Signaling Technology, Beverly, MA, USA). Band densities on autoradiograms were densitometrically quantified (Quantity One software; Bio-Rad), with GAPDH serving as the internal control.

The experimental procedure for Western blotting analysis that was used in this study was followed as described in our previous study [29 (link)]. The horseradish peroxidase-conjugated IgG that was anti-mouse and anti-rabbit (Thermo Fisher Scientific, New York, NY, USA) was used in this study. GAPDH was used as the control and for quantification. Antibodies purchased from Santa Cruz, USA: anti-EPCAM (1:1000, sc-25308), anti-ALDH1 (1:300, sc-374149), anti-ALDH2 (1:300, sc-100496), anti-KLF4 (1:1000, sc-166100), anti-GAPDH (1:1000, sc-47724). Antibodies purchased from Cell Signaling Technology, USA: anti-SNAI2 (#9585, 1:1000), anti-SOX2 (#3579, 1:1000), anti-OCT4 (#2840, 1:1000), anti-NANOG (#4903, 1:1000), anti-β-catenin (#8480, 1:1000), anti-c-Myc (#18583, 1:1500 dilution). Anti-PCNA (#60097-1-Ig, 1:5000) were purchased from Proteintech. The signal intensity was quantified using the protein imprinting imaging system (Tanon 5200, Better Tanon, Shanghai, China).