Anti mdr1

The total protein was extracted from cells lysed with sodium dodecyl sulfate (SDS, Beyotime Biotechnology, Shanghai, China) lysate containing 1% phenylmethanesulfonyl fluoride (Beyotime Biotechnology) and 1% phosphatase inhibitor. The cytoplasmic and nuclear proteins were isolated with the Minute™ Cytoplasmic & Nuclear Extraction Kit (#SC-003; Invent Biotechnologies, Inc, Minnesota, USA) according to the manufacturer's instructions. The protein concentration was measured with the BCA protein assay kit (Beyotime Biotechnology). Protein samples were run on SDS-polyacrylamide gel electrophoresis (SDS- PAGE) and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was incubated with the primary antibody at 4 ℃ overnight. The following antibodies were used in the study: anti-CDK14 antibody (1:1000 dilution, Santa Cruz Biotechnology, Inc, Dallas, Texas USA), anti-MDR1 (1:5000 dilution, P-gp, Cell Signaling Technology, Inc., Boston, MA, USA), and anti-β-actin (1:5000 dilution, Proteintech, Wuhan, China). The secondary antibodies of the anti-mouse IgG or anti-rabbit IgG were used at RT for 1 h. The protein bands were photographed by the chemiluminescence imaging system (Tanon Science & Technology, Shanghai, China).

Total cellular protein was extracted from 2D cultures as previously described [31 (link)]. Protein concentration was determined by Lowry assay and an equal concentration of protein extracts (40 µg) was loaded on SDS-PAGE gels, transferred onto a nitrocellulose membrane and immunoblotted with primary antibodies overnight. Anti-EDB-FN antibody (G4 clone) was used at 1:1000 dilution. The following primary antibodies (1:1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA): anti-E-cadherin (Cat#3195), anti-Slug (Cat#9585), anti-phospho-T308-AKT (Cat#13038), anti-phospho-S473-AKT (Cat#4060), anti-pan-AKT (Cat#4691), anti-MDR1 (Cat#12683S); and anti-Histone H3 (Cat#4499) and anti-β-actin (Cat#4970) as loading controls. The anti-Phosphoepitope SR proteins (Cat#MABE50; clone 1H4) and anti-SRp40 (Cat#06-1365) antibodies were purchased from Millipore Sigma (Temecula, CA, USA) and used at 1:500 dilution. Anti-N-Cadherin antibody (Cat#76057) was purchased from Abcam (Cambridge, MA, USA) and used at 1:500 dilution. The background-adjusted pixel intensities of test proteins were normalized with those of actin controls in FIJI, and the levels were expressed as ratio of treated over non-treated cells near the respective lanes.