Odyssey clx system

Cells were lysed in Radioimmunoprecipitation (RIPA) assay buffer (Sigma, St. Louis, MO), total protein was extracted, and the protein concentration was measured by Bradford Protein Assay (Bio-Rad). Protein samples were loaded on 10% SDS-PAGE gel and the separated proteins were transferred by electroblotting to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with Odyssey^® Blocking Buffer (LI-COR Biosciences, Lincoln, NE) and incubated with the primary antibody and secondary antibody. Immunolabeling was detected by using Odyssey^® CLx system (LI-COR Biosciences). The following primary antibodies were used: Anti-Hydroxyproline antibody (ab37067, Abcam; 1:1000 dilution); Anti-P4HA1 antibody (ab59497, Abcam; 1:500 dilution); Anti-P4HA2 antibody (A4262, ABclonal Inc.; 1:500 dilution); Anti-GAPDH antibody (sc-32233, Santa Cruz Biotechnology, Inc.; 1:1000 dilution).

RNA-EMSAs were performed as described elsewhere [36 (link)]. Briefly, RNA oligos were ordered corresponding to the mature form of miR-130a-3p (5’-CAGUGCAAUGUUAAAAGGGCAU-3’), a 21-mer sequence of the Jarid2 3’-UTR (5’-Auagcuacccacauugcacug-3’) containing the target site for miR-130a and a scrambled control (5’-GGuuAACuCGCCAAuGAuCCu-3’) from IDT. The miR-130a-3p sequence was labeled with 5’-IRDye 700. Labeled miR-130a-3p probes (200 nM) were incubated with the corresponding target or scrambled RNA molecules in the presence of 10 mM MgCl₂, 100 mM NaCl, 50 mM Hepes pH 7.2 and 5% glycerol for 30 minutes at 37°C. Binding reactions were run in a 12% polyacrylamide gel for 3 h at 120 V (4° C) in 1X TBE. Gels were scanned using a LI-COR Odyssey CLx system and assembled.

Tumor samples were collected after surgery, frozen immediately in liquid nitrogen, and stored at −80°C. Proteins were extracted and electroblotted onto nitrocellulose membranes as already described [20 (link)]. Immunoblot analysis was performed on normal and cancerous MTC tissues. Briefly, membranes were incubated overnight with the following primary antibodies: anti-PTTG1 (GeneTex, Irvine, USA), anti-Aurora kinase A (Cell Signaling Technology, Danvers, USA), and anti-β-actin (GeneTex, Irvine, USA). Then, secondary antibodies were added for 1 h with anti-mouse and anti-rabbit (1 : 800) secondary IRDye. Membranes were scanned with the Odyssey CLx system (LI-COR BioSciences, Milan, Italy) equipped with an infrared light technology for the detection. Signal intensity was quantified with Image Studio™ software (Version 4.0, LI-COR BioSciences) following the manufacturer's instructions. The experiments were performed three times.

CK cells were mock infected or infected with the indicated viruses, and at 24 h postinfection, cells were scraped into the culture medium and pelleted. Cell pellets were washed once with cold PBS and then lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris [pH 7.4], 1% Igepal, 0.25% sodium deoxycholate, 1 mM sodium orthovanadate, 1× protease inhibitor cocktail) on ice for 20 min. Cell lysates were mixed with 4× solubilization buffer (SB; Bio-Rad) to give a 1× final concentration and denatured at 95°C for 3 min. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking overnight in 5% milk in PBS-Tween 20, the membrane was labeled with anti-S2 (catalog number 26.1; Thermo Fisher) and anti-beta-actin (Abcam), followed by IRDye 680- and 800-labeled secondary antibodies (Li-Cor). Blots were imaged using an Odyssey CLx system (Li-Cor).

Pericytes were incubated for 48 h with 50 ng/ml FGF-2 in DMEM containing 2% FBS prior to stimulation with 10 ng/ml PDGF-BB for 10 min. For time-course experiments, cells were treated for 10 min, 1 and 6 h with 50 ng/ml FGF-2, 50 ng/ml PDGF-BB, or a mixture of FGF-2 and PDGF-BB. Non-treated pericytes were used as a control. Soluble proteins from total cell lysates were applied to an SDS-PAGE (NP0301; Invitrogen), followed by wet transferring onto a methanol-activated polyvinylidene fluoride membrane (LC2002, Invitrogen). Membranes were blocked at room temperature with 3% skim milk for 60 min, followed by incubation overnight with a rabbit anti-mouse PDGFRβ (1:1000; 28E1; Cell Signaling) antibody, a rabbit anti-mouse phospho-PDGFRβ antibody (1:1000; G63G6; Cell Signaling), or an anti-mouse β-actin antibody (1:1000; 3700S; Cell Signaling). Membranes were incubated at room temperature for 60 min with a mixture of secondary antibodies consisting of an anti-mouse secondary antibody conjugated with IRDye 680RD (1:15,000; LI-COR; Lincoln) and an anti-rabbit secondary antibody conjugated with IRDye 800CW (1:15,000; LI-COR; Lincoln). Protein signals were captured using an Odyssey CLx system (LI-COR). Full-gel images are shown in Supplementary Fig. S6.