Nupage gradient gel

Identical amounts of total protein were heat-denatured and reduced (70°C; 10min) then submitted to SDS-PAGE separation on 4–12% gradient NuPAGE gels (Life Technologies, Saint Aubin, France) and transferred to polyvinylidene fluoride membranes. The membranes were blocked for 1 hour in 5% BSA solution and incubated with the appropriate primary and HRP-conjugated secondary antibodies (1:1,000 and 1:10,000 dilution, respectively). Immunodetections were performed using ECL reagent and image acquisition was performed by using a chemiluminescent CCD imager ImageQuant LAS 4000 (GE Healthcare, Velizy-Villacoublay, France). Densitometric analysis of the bands was performed with the ImageQuant TL software (GE Healthcare). MAST2 antibody (ab209079) was from Abcam, PTEN (138G6), AKT (C67E7), phospho-AKT Ser473 (193H12) and β-actin (13E) antibodies PTEN were from Cell Signaling Technology.

Identical volumes of microparticle-free CM (serum free) and plasma or identical amounts of total protein were heat-denatured and reduced (70 °C; 10 min) then submitted to SDS-PAGE separation on 4–12% gradient NuPAGE gels (Life Technologies, Saint Aubin, France) and transferred to polyvinylidene fluoride membranes. Membranes were blocked for 1 h in 5% BSA solution and incubated with the appropriate primary and HRP-conjugated secondary antibodies (1:1000 and 1:10,000 dilution, respectively). Immunodetections were performed using ECL reagent and image acquisition was performed by using a chemiluminescent CCD imager ImageQuant LAS 4000 (GE Healthcare, Velizy-Villacoublay, France). Densitometric analysis of the bands was performed with the ImageQuant TL software (GE Healthcare, Velizy-Villacoublay, France).

Total protein was extracted in RIPA buffer supplemented with protease and phosphatase inhibitors (Beyotime, JiangShu, China). Protein concentration was determined using the BCA protein concentration determination kit (Beyotime, JiangShu, China). Protein from the control and treated cell lysates was loaded onto 8-12% gradient NuPAGE gels (Novex, San Diego, CA, USA), electrophoresed under reducing conditions, and transferred onto polyvinylidene difluoride membranes (0.22 μm; Millipore, Billerica, USA). Western blot analysis was performed as described previously [25 (link)]. ASCL2 Monoclonal Antibody (MAB4417, Millipore, Billerica, USA), E-cadherin, vimentin, MMP2, MMP9, Bcl-2, Bax, Caspase-3 and GAPDH antibody (Cell Signaling Technology, MA, USA) were used.

The western blotting assay was performed to evaluate the endogenous AMACR expression and the efficiency of AMACR knockdown in GIST882 and GIST48 cell lines. Cell lysates containing 25 μg protein were separated by 4-12% gradient NuPAGE gel (Invitrogen), transferred onto PVDF membranes (Amersham), and probed with antibodies against GADPH (1;3000, Chemicon), and proteins of interest. The latter included anti-AMACR (1:250, Invitrogen), anti-cyclin D1 (1:10000, Epitomics), anti-cyclin E (1:200, Abcam), anti-CDK2 (1:2000, Epitomics), and anti-CDK4 (1:200, Abcam). After incubation with the secondary antibody, proteins were visualized by the chemiluminescence system (Amersham).

The western blotting assay was performed based on that of our previous publications [45 (link), 46 (link)] to evaluate the endogenous MCM10 expression and the efficiency of MCM10 knockdown in J82 and TCCSUP cell lines. Cell lysates containing 25 μg protein were separated by 4-12% gradient NuPAGE gel (Invitrogen, Carlsbad, CA), transferred onto PVDF membranes (Amersham, Biosciences, Buckinghamshire, UK). After blocking with 5% skimmed milk in TBST buffer at room temperature for 1 h, the membranes were then probed with antibodies at 4°C overnight against MCM10 (1:1000, H-41, Santa Cruz), and GAPDH as a loading control (6C5, 1:10,000, Millipore, Beverly, MA). After incubation with the secondary antibody at room temperature for 1.5 h, proteins were visualized by the chemiluminescence system (Amersham Biosciences).

For the size-exclusion chromatography (SEC) assays in Figure 2B, purified Pab1 (36 μM final concentration in the case of 90A, 24 μM in the case of 70A and either 36 μM (“3x 30A/Pab1”), 24 μM (“2x 30A/Pab1”) or 12 μM (“Pab1” and “Pab1/30A”) final concentration in the case of 30A was mixed with the respective RNA (90A oligo RNA at 12 μM final concentration, 70A oligo RNA at 12 μM, 30A oligo RNA at either 36 μM [“3x 30A/Pab1”], 24 μM [“2x 30A/Pab1”] or 12 μM [“Pab1/30A”]) final concentration and incubated at 4°C for 30 min (all oligo(A) RNAs were procured from Ella Biotech). Pan2cat-Pan3 (12 μM final concentration) was added to these reactions and again incubated for 15 min at 4°C in a total injection volume of 30 μl in SEC buffer (20 mM HEPES/NaOH pH 7.5, 50 mM NaCl, 100 mM potassium acetate, 5 mM magnesium acetate, 2 mM DTT). Complex formation was assayed by comparing the retention volumes in SEC on a Superose 6 3.2/300 column (GE Healthcare). Composition of the SEC peak fractions were analyzed by SDS-PAGE on 4%–12% NuPAGE gradient gels (Thermo Fisher Scientific) and visualized by Coomassie staining. For better comparison, each individual chromatogram was normalized by its maximal absorption value and plotted in R using the tidyverse collection of R packages.