Flag f1804

Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans-replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

Cells were harvested and lysed in lysis buffer (1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 50 mM Tris pH 7.5, 0.15 M NaCl, 50 mM NaF, 1 mM EDTA, 1 mM DTT, 1 mM Na₃VO₄) with proteasome and phosphatase inhibitors. Following a 30-min incubation on ice, the supernatant was collected by centrifugation at 13,600 rpm for 25 min at 4 °C. Protein samples were separated by SDS polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and incubated with the appropriate primary and secondary antibodies. Proteins were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo, 34,580). The following primary antibodies were used: TOP1 (ab3825, Abcam), cyclin B1 (12231#), caspase-3 (9665#), cleaved caspase-3 (9661#), PARP (9542#), cleaved-PARP (5625#), p-CHK1 (S345) (2348#), P21 (2947#) (Cell Signaling Technology), CUL 1 (sc-17775) and t-CHK1 (sc-8408) (Santa Cruz), actin (A5441#) and FLAG (F1804) (Sigma).

TOPK (sc-293028) was purchased from Santa Cruz Technology, Inc (Santa Cruz, CA). Flag (F1804) was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies to detect ALK (#3633), p-ALK (#3341), p-JNK (#4668), JNK (#9258), p-ATF2 (#5112), ATF2 (#9226), PARP (#9542), c-PARP (#5625), HA (#3724), p-Tyrosine (#8954) were purchased from Cell Signaling Technology (Danvers, MA). β-actin (66009-1-Ig),α-Tubulin (66031-1-Ig), HRP-labeled Goat anti-Mouse IgG (H + L) and Goat anti-Rabbit IgG (H + L) were from Proteintech Group, Inc (Wuhan, HB). Phospho-TOPK at Y74 antibody was prepared by Abgent, Inc (Suzhou, JS). All antibodies were used following the instructions of the respective manufacturers.

For western blotting analyses, whole-cell lysates were prepared and 20 µg of proteins were resolved on 12% SDS–PAGE, transferred onto nitrocellulose membranes (Schleicher & Schuell Bioscience, Dassel, Germany) and probed with antibodies for PTEN (sc-7974; Santa Cruz Biotechnology, Santa Cruz, CA) (1:1000), APE1^{14 (link)} (1:1000), FLAG (F1804, SIGMA) (1:5000), Akt1 (sc-1618, Santa Cruz Biotechnology) (1:1000) and p-Akt1/2/3 (Ser473)(sc-7985-R; Santa Cruz Biotechnology) (1:1000). Data normalization was performed by using monoclonal anti-actin and anti-tubulin (Sigma-Aldrich) as indicated. The corresponding secondary antibodies labeled with IR-Dye (anti-rabbit IgG IRDye 680 and anti-mouse IgG IRDye 800) were used. Detection and quantification was performed with the Odyssey CLx Infrared imaging system (LI-COR GmbH, Germany). The membranes were scanned in two different channels using an Odyssey IR imager; protein bands were quantified using Odyssey software (Image Studio 5.0) and the relative signal, expressed as ratio of the treated group over the control group, was calculated.

Matrigel was purchased from BD Bioscience (Bedford, MA, USA). ShRNA constructs selectively targeting NDUFS6, NDUFA7, NDUFA9, and NDUFA11 were purchased from Sigma Aldrich (MISSION shRNA library, St. Louis, MO, USA). Recombinant human IL-6 was purchased from Peprotech (Cranbury, NJ, USA; 200-06). NAC (A7250) was purchased from Sigma Aldrich (St. Louis, MO, USA). Antibody CD16/32 (101302, clone:93) was purchased from BioLegend (San Diego, CA, USA). Antibodies F4/80 (1950719, clone: BM8), CD11c (2011154, clone: N418), CD45 (2055168, clone: 104), CD11b (2011193, clone: M1/70), CD86 (1987724, clone: GL1), and CD206 (2073756, clone: MR6F3) were purchased from Invitrogen (Waltham, MA, USA). Antibody RORα (E0713) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies Flag (F1804) and tubulin (AB9354) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibody NDUFA11 (17879-1-AP) was purchased from Proteintech.