Sf3b4

Four-micrometer-thick sections were used for IHC staining. The sections were deparaffinized and rehydrated with xylene and a graded series of ethanols. Then, the slides were incubated using the primary antibodies (SF3B4, Proteintech, 10482–1-AP and Ki-67, Cell Signaling Technology, 9449T) at 4 °C overnight after antigen retrieval. Finally, a DAB detection system (ZSGB-BIO, Beijing, China) was used to detect staining.

Proteins were separated by 8% SDS–PAGE and transferred to a PVDF membrane. The membranes were blocked and incubated first with primary antibodies at 4°C overnight and then with horseradish peroxidase–conjugated secondary antibody. Antibodies against NCL, hnRNPD, hnRNPC, hnRNPK, SF3B4, IGF2BP1, RBM3, MGMT, hnRNPUL1, polyadenylate‐binding protein 1 (PABPC1), ZEB‐1 and AGO2 were obtained from Proteintech (Wuhan, China). Antibodies against GAPDH, AKT, phospho‐AKT (T308), phospho‐AKT (Y326) and phospho‐AKT (S473) were obtained from Bioworld Technology, Inc. (MN, USA). Antibody against IMPAD1 was obtained from Abcam (Cambridge, UK). Antibody against hnRNPCL1 was obtained from Abgent (Taiwan, China). Antibodies against E‐cadherin, Src and U1 SnRNP70 were obtained from Santa Cruz (CA, USA). Antibodies against N‐cadherin, vimentin, Snail, and Slug were obtained from GeneTex (CA, USA). Antibodies against phospho‐Src (T416), PDPK1, phospho‐PDPK1 (S241), mTOR, phosphor‐mTOR (S2448) EGFR, Phospho‐EGFR (Tyr1173), VEGFR and Phospho‐VEGFR (Tyr951) were obtained from Cell Signaling Technology (Danvers, MA, USA). The densities of developed protein bands were analysed using TotalLab v2.01 (Nonlinear Dynamics Ltd).

The protein was extracted from the cultured cells and frozen tissue samples using RIPA lysis buffer and protease inhibitor cocktail as described previously [30 (link)]. The same amount of protein was loaded onto the gel using the modified Bradford method for protein quantitative analysis. After being separated by SDS-PAGE, the proteins were electrotransferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010). The membrane was blocked with 5% nonfat milk for 2 h. Finally, the membranes were incubated with the primary antibody overnight at 4 °C. The antibodies used were as follows: SF3B4 (Proteintech, 1:1000, 10482-1-AP), MMP1 (Proteintech, 1:1000, 10371-2-AP), E-cadherin (Proteintech, 1:500, 20874-1-AP), vimentin (Proteintech, 1:1000, 10366-1-AP), Twist1 (Proteintech, 1:1000, 25465-1-AP), ZO-1 (Proteintech, 1:500, 21773-1-AP), KLF16 (Abcam, 1:500, ab187973) and β-actin (Cellsignal, 1:1000, sc-47778). After reaction with HRP-labeled secondary antibody (1:10000, Rockland), the membrane was treated with Immobilon™ Western chemiluminescence HRP substrate (Millipore) and detected by ECL (enhanced chemiluminescence) Fuazon Fx (Vilber Lourmat).