β actin

Western blotting was performed as described previously^{48 (link)}. Following quantification of protein concentration, 30 µg of protein samples were boiled in gel-loading buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and probed with the following primary antibodies: β-actin, α-tubulin, GSK-3 (BD Transduction laboratories, Oxford, UK), AT100 and Tau-1 (Innogenetics, Ghent, Belgium), GAPDH, GSK-3β, and P9Ser-GSK-3β (Cell Signaling, Leiden, Netherlands), and synaptophysin (Abcam, Cambridge, UK). Next, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Isis Ltd., Bray, Ireland) and protein bands visualized using chemiluminescence (Pierce Biotechnology, Rockford, IL, USA). Gel bands were captured using a Fujifilm LAS-3000 (Fujifilm, Tokyo, Japan) and analyzed using Alpha-EaseFC4.0 software.

Western blot analysis was performed as previously described [53 (link)]. Antibodies against the following proteins were used for immunoblot: MCM2 (ab108935) and phospho-MCM2 (ab133243) (Abcam), p27 (610242; BD Transduction Laboratories), β-actin (4970), c-Myc (5605), phospho-Ser345-Chk1 (2348), cleaved PARP (5625), γ-H2AX (2577), GAPDH (2118), p21 (2947), STAT1 (9175), and phospho-Ser727-STAT1 (8826) (Cell Signaling Technology), phospho-Ser10-histone H3 (06–570; Millipore), Cdc6 (sc-9964), CDK8 (sc-1521), Cyclin B1 (sc-752), and FANCD2 (sc-20022) (Santa Cruz Biotechnology), and α-tubulin (T9026) and CDK19 (HPA007053) (Sigma-Aldrich). α-tubulin, β-actin, and GAPDH were used as loading controls.

Whole-cell proteins from PBLs were extracted in RIPA buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 10% Glycerol, 1% NP40, 2 mM EDTA) and cellular debris were eliminated after centrifugation at 12,000 g for 25 min at 4°C. Protein concentration was assessed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Fifteen microgram of proteins were diluted in Laemmli buffer, separated on polyacrylamide gels (NuPAGE 4–12% Bis-Tris Gel, Life Technologies) and transferred to a PVDF membrane (Immobilon-P, Merck-Millipore, Darmstadt, Germany). Transfer efficiency was evaluated with Ponceau staining. Next, the membrane was saturated using 5% non-fat milk diluted in TBS 0.05% Tween (TBS-T) for 1 h, incubated with mouse anti-human MYB monoclonal antibody (2 μg/mL, #05-175, Merck-Millipore) overnight at 4°C. After several washes in TBS-T, the membrane was incubated with HRP sheep anti-mouse secondary antibody (#NA931, GE Healthcare, United Kingdom) for 1 h at room temperature. Finally, the membrane was washed in TBS-T and revealed with electrochemiluminescence (ECL) on autoradiography films (Amersham, GE Healthcare). MYB intensity was normalized on β-actin (#612656, BD Transduction Laboratories) expression. Band intensity quantification was done using ImageJ software.

Western blotting was performed as described previously [26 (link)]. The primary antibodies used in this study and their dilutions were as follows: ROR1 (1/500, homemade IC5 or 5B3 clones), β-actin (1/5000), E-cadherin (1/1000, BD Transduction Laboratories), Vimentin (1/1000), PARP (1/1000, Cell Signaling), CK19 (1/1000, Santa Cruz Biotechnology), and His-tag (1/3000, Qiagen). After treatment of PVDF membranes (Thermo Fisher Scientific) with primary antibodies, HRP-conjugated secondary antibody (1/3000, Cell Signaling) and Amersham ECL Select (GE Healthcare) chemiluminescence substrate were used to visualize protein bands by using the ChemiDoc XRS system (Bio-Rad). RNA isolation, cDNA synthesis, and RT-qPCR were performed as described before [26 (link)]. Relative expression of ROR1 mRNA in HCC cell lines was measured by normalizing ROR1 expression to that of GAPDH and calculated with the 2^{− ΔCt} formula [ΔCt =Ct (ROR1) − Ct (GAPDH)]. Primers for RT-qPCR were designed using Primer-BLAST. Sequence of primers were as follows: ROR1-F 5′-GTTTCCCAGAGCTGAATGGA-3′ and ROR1-R 5′-GGATGTCACACAGATCAGACTT-3′; GAPDH-F 5′-GGCTGAGAACGGGAAGCTTGTCAT-3′ and GAPDH-R 5′-CAGCCTTCTCCATGGTGGTGAAGA-3′.

MDMs and moDCs were infected with ZIKV^PR (MOI=1.0) at 37°C. At 48hpi, the cells were untreated or treated with 1000U/mL of recombinant human IFN-α for 30 min and then lysed with RIPA buffer supplemented with Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail II (Thermo Fisher Scientific). The expression levels of STAT1, STAT2, STAT1 phosphotyrosine residue 701 (pSTAT1), STAT2 phosphotyrosine residue 689 (pSTAT2), and β-actin were detected using anti-STAT1 (BD Transduction Laboratories), anti-STAT2 (SANTA CRUZ BIOTECHNOLOGY), anti-pSTAT1 (Cell Signaling Technology), anti-pSTAT2 (Millipore), and anti-β-actin (Sigma-Aldrich) primary antibodies. The intensity of the bands was calculated using Image J software to generate the histogram. For evaluation of knockdown efficiency of siRNA transfection, MDMs and moDCs were transfected with scrambled siRNA or siRNA targeting STAT1 or STAT2. The expression of STAT1, STAT2, and β-actin were detected by Western blots.

The protein was extracted from the lung adenocarcinoma cells using Radio-Immunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime) at 4 °C. Protein concentrations were measured by the Bicinchoninic Acid Protein Assay (BioRad, Hercules, CA, USA). Equal amounts of whole-cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) followed by incubation with primary mouse monoclonal antibodies against human AIB1 (1:1000 dilution), CXCR4 (1:500 dilution), tumor necrosis factor (ligand) superfamily member 10 (TNFSF10) (1:500 dilution), matrix metallopeptidase 11 (MMP11) (1:1000 dilution), matrix metallopeptidase 2 (MMP2) (1:500 dilution), and vascular endothelial growth factor A (VEGFA) (1:1000 dilution) (BD Transduction Laboratories) overnight at 4 °C. β-Actin was used as an internal control (1:1000 dilution, BD Transduction Laboratories). After washing, the polyvinylidene fluoride (PVDF) membranes were incubated with secondary antibody (goat anti-mouse, 1:10,000 dilution, Cell Signaling Technology, Danvers, MA, USA) for 2 hat room temperature. The immunoreactive proteins were detected with enhanced chemiluminescence detection reagents (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s instructions.