Pvdf membrane

For PrP analysis in brain extracts, brain homogenates (3 different animals) prepared in PBS were either not digested or treated with different concentrations of PK (0 to 5 mg/ml; VWR, Ca) as indicated for one hour at 37°C. The reaction was terminated by adding 1X pefabloc proteinase inhibitor (VWR, Ca). Fifty μg of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred to PVDF membranes (Millipore, Ca). PVDF membranes were probed using anti-PrP monoclonal antibodies followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma, Ca) and developed using ECL-plus detection (Amersham). Images were acquired on X-ray film (Super Rx; Fujifilm) or by using a digital imaging system (Alpha Innotech, FluoriChemQ). FluoChemQ software (Alpha Innotech) was used to quantify and determine the relative values of PrP^res signals.

Protein was extracted from PC9, PC9GR, HCC827 cells using RIPA Lysis Buffer with protease and phosphatase inhibitors (P0013B; Beyotime; China). BCA Protein Assay kit was used to determine the protein concentration (P0012S; Beyotime; China). Cell lysate was separated by SDS-PAGE electrophoresis and then transferred to PVDF membranes (Millipore). Next, the PVDF membranes was incubated with primary antibodies and followed by horseradish peroxidase-conjugated secondary antibody (Sigma) after blocking with PBST containing 5% non-fat milk. The protein signals were visualized by enhanced chemiluminescence ECL reagent (Thermo Scientific Pierce). GAPDH was used as a loading control.

The sodium iodide symporter (NIS) is a cell membrane protein located in the basolateral membrane of thyroid follicular cells and mediates iodine transport in the cells. The relative level of NIS was detected by Western blotting using its antibody (abcam, Cambridge, UK. Catalog No. ab199410). GAPDH (Cell Signaling Technology. Catalog No. 2118) was used as an internal reference.
Rat thyroid or human Nthy cells were pretreated with RIPA lysis buffer and protease inhibitors (Beyotime Biotechnology. Catalog No. P0013 and P1005) to extract total proteins. Total proteins were determined using a multimode microplate reader (type BioTek Synergy H1, Agilent, Inc.) and separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes (Merck Millipore, MA, USA. Catalog No. IPVH00010). The PVDF membranes were blocked with BSA (Sigma-Aldrich. Catalog No. 9048-46-8) and then hybridized with antibodies. Finally, the PVDF membranes were incubated with a developing solution and developed in the Molecular Imager Gel Doc XR system (Bio-Rad Laboratories, Inc.). Western blots were analyzed with ImageJ software (National Institutes of Health).

Cells were harvested at 48 h post transfection, and lysed in SDS lysis buffer (Beyotime, Beijing, China) with fresh addition of 1% protease inhibitor cocktail (Sigma-Aldrich, Darmstadt, Germany) and 1 mM PMSF (Sigma-Aldrich). Protein concentrations were determined by the Bradford method with the BCA Protein Assay kit (Thermo Fisher Scientific). Equal amount of proteins (30 µg) were resolved in 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking, PVDF membranes were washed and incubated with different primary antibodies [1:1000 diluted mouse anti-BCCIP (08269, Sigma-Aldrich), rabbit anti-BNIP3L (12,396, Cell signaling technology, Danvers, MA, USA), rabbit anti-SOX2 (3579, Cell signaling technology), goat anti-MST1 (AF4949, R&D systems, Minneapolis, MN, USA), goat anti-EFNA4 (AF369, R&D systems) antibodies, 1:5000 diluted rabbit anti-α-tubulin monoclonal antibody (2125, Cell signaling technology)] at 4 °C overnight. After re-warming for 30 min at room temperature and three times washing, membranes were incubated with HRP-linked goat anti-rabbit, goat anti-mouse, or rabbit anti-goat IgG (1:5000 diluted, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 37 °C. Protein bands were developed by using the ECL kit (Tiangen Biotech, Beijing, China), and images were captured with Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

Total tissue and cell were harvested and then lysed in RIPA buffer (50 mM Tris pH 7.5, 0.5% sodium deoxycholate, 150 mM NaCl, 10 mM NaF, 0.1% SDS, 1% Triton X-100) added protease inhibitors (Roche, Switzerland). The lysates were centrifuged at 12,000 g for 25 min at 4°C. Then, the concentration of protein was examined by the BCA Protein Assay kit (Genstar, China). Protein samples were separated by 10% SDS-PAGE after incubation at 95°C for 15 min in SDS sample buffer, and then transferred to PVDF membranes (Millipore, Boston, MA, U.S.A.). Next, the membranes were blocked with 5% (w/v) evaporated milk in TBST for 1 h at 25°C. The blocked membranes were put into TBST solution that contains primary antibodies (anti-FBXW7, anti-STYX and anti-GADPH) at 4°C overnight, and then washing five times with TBST solution. The PVDF membranes were incubated for 1 h at room temperature in IgG horseradish peroxidase secondary antibody (Sigma-Aldrich). After washing three times with TBST, they were imaged using StarSignal Plus Chemiluminescent Assay Kit (Genstar, China).

After transfection for 48 hours, western blot assay was carried out using the standard method, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). After 2 hours of blocking, PVDF membranes were incubated with anti‐mouse β‐actin antibodies (Sigma‐Aldrich) and anti‐rabbit EIF3a antibodies (Cell Signaling Technology, USA) overnight at 4°C.

Whole-cell lysates of HCT116 and SW480 cells were extracted using lysis buffer, including 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were centrifuged at a speed of 14000 rpm for 15 min, then mixed with 5×loading buffer and denatured at 100 °C for 5 min. Aliquots of supernatant protein (40 ~ 50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The PVDF membranes were then blocked for 1 h with 5% (w/v) non-fat milk at room temperature. After incubation with anti-PRMT5 (Abcam, ab109451), anti-EZH2 (ProteinTech, 21800-1-AP), anti-CDKN2B (Abcam, ab53034) or anti-GAPDH (ProteinTech, 60004) overnight at 4°C, the PVDF membranes were washed with PBST and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich) at room temperature. Subsequently, the immunoreactive bands were visualized using ECL detection reagent (Thermo Scientific).