Anti myc tag

HEK293 cells were transfected in 6-well plates and cultured for 48 h. Cells were lysed for 10 min at 4 °C with 100 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.2% Triton X-100, 1% PMSF, and protease inhibitor cocktail. Cell lysates were cleared by centrifugation at 10,000×g for 3 min at 4 °C. Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to PVDF membranes using a TransBlot Turbo Blotting apparatus (Bio-Rad). Membranes were blocked in PBS containing 5% milk and 0.1% Tween-20 and incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-GFP (Clontech cat. no. 632380, 1:8000, for YFP constructs); anti-Myc tag (Abcam cat. no. ab9106, 1:1000); and anti-β-actin (Sigma cat. no. A5441, 1:10,000). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 45 min at room temperature. Proteins were visualized using Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) and a ChemiDoc XRS+ imaging system (Bio-Rad).

Total protein extracts were obtained using the lysis buffer (120 mM NaCl, 20 mM Tris-HCl pH 7.5, 2% Nonidet P40) completed with a mix of proteases and phosphatases inhibitors. 80 μg of extracted proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked with BSA or 5% not-fat milk and then incubated with the following antibodies: anti-myc tag (ab9132, Abcam, Cambridge, UK), anti-MPPED2 (H00000744-D01P, Abnova, Taipei City, Taiwan), anti-PTEN (ab32199, Abcam), anti-pospho-Akt (Ser473) (#4051, Cell Signaling, Danvers, MA, USA), anti-Akt (#92725, Cell Signaling). To normalize the amount of protein loaded, the membranes were incubated with anti-GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-β-Actin protein (Sigma-Aldrich). Filters were then incubated with horseradish peroxidase-conjugated secondary antibody (1:3000) for 1 h at room temperature and the signals were detected by western blotting detection system (ECL).

HEK293T cells were seeded in 6-well plates and transfected with the indicated plasmids. After 24-h incubation, cells were scraped and resuspended in Pierce IP Lysis Buffer (Thermo Fisher Scientific, USA) supplemented with protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation (16,000 ×g, 4°C, 15 min). Antibodies were added and incubated with 50 μl protein A magnetic beads (SureBeads, Bio-Rad, USA) with rotation. The beads were washed extensively with PBS supplemented with 0.1% Tween 20. Beads probed with appropriate antibodies were incubated with 300 μg proteins 4°C with rotation. Proteins were analyzed by immunoblot analysis. Protein samples were probed with the following primary antibodies: anti-FLAG tag (1:2000; Fujifilm Wako Pure Chemical Corporation, Japan), anti-MYC tag (1:2000; Abcam, UK)

RIPA buffer (Sigma-Aldrich) plus a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) was used to lyse cells as previously described [18 (link)]. Proteins were loaded onto SDS-PAGE gels, transferred onto polyvinylidene difluoride membranes, and blocked with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at 4 °C. The following antibodies were used; Anti-FLAG tag (Wako), Anti-MYC tag (Abcam), Anti-GFP (Santa Cruz), Anti-Tubulin or β-actin antibodies (Abgent). Anti-pSTAT1/STAT1, Anti-HA tag, Anti-HIS tag, Anti-MYC tag, Anti-MDA5, Anti-RIG-I, Anti-pTBK1/TBK1, Anti-pIRF3/IRF3, Anti-MxA, Anti-OAS1, and Anti-RNaseL antibodies were from Cell Signaling Technology. Anti-SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (Sino Biological), while Anti-IFV NP antibody and Anti-ZIKV E antibody were purchased from GeneTex. The blots were washed with TBST for three times and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Proteins were visualized with ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Densitometry to quantify immunoblot bands was performed using ImageJ software.

Anti‐Arf1 antibody (#10790‐1‐AP and #20226‐1‐AP), anti‐Rab35 antibody (#11329‐2‐AP), anti‐HDAC6 antibody (#16167‐1‐AP), anti‐NAT10 antibody (#13365‐1‐AP), anti‐Ran antibody (#10469‐1‐AP), anti‐TPX2 antibody (#11741‐1‐AP), anti‐Myt1 antibody (#67806‐1‐lg), anti‐GAPDH antibody (#60004‐1‐lg), and anti‐α‐tubulin antibody (#11224‐1‐AP) were purchased from Proteintech (Rosemont, IL, USA). Anti‐GM130 antibody (#DF7556) was purchased from Affinity Biosciences (Cincinnati, OH, USA). Anti‐Aurora A antibody (#AF1708) was purchased from Beyotime (Shanghai, China). Anti‐Plk1 antibody (#37‐7000) was purchased from Invitrogen (Carlsbad, CA, USA). Anti‐Ac‐tubulin antibody (#T7451), anti‐α‐tubulin‐FITC antibody (#F2168), and Hoechst 33 342 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti‐Myc tag (#ab18185), Alexa Fluor 594 Goat Anti‐Rabbit IgG (H + L) (#ab150080), Alexa Fluor 594 Goat Anti‐Mouse IgG (H + L) (#ab150116), anti‐CDK1 antibody (#ab18), anti‐cyclin B1 antibody (#ab181593), and Anti‐GM130 antibody (#ab52649) were purchased from Abcam (Cambridge, UK). Anti‐phospho‐Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) antibody (#2914T) and antiactin antibody (#3700) were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase–conjugated goat antirabbit/mouse antibodies (CW0103/CW0102) were purchased from CWBIO (Beijing, China).

HEK293 cells were transfected in 6-well plates and cultured for 48 h. Cells were lysed in 300 μL of Laemmli sample buffer containing 10% Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and incubated for 10 min at 95 °C. Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to PVDF membranes using a TransBlot Turbo Blotting apparatus (Bio-Rad). Membranes were blocked in PBS containing 5% milk and 0.1% Tween-20 and incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-GFP (Clontech cat. no. 632380, 1:8000, for YFP constructs); anti-mCherry (Novus cat. no. NBP1-96751, 1:1000); anti-V5 tag (Genetex cat. no. GTX42525, 1:3000); anti-Myc tag (Abcam cat. no. ab9106, 1:1000); anti-β-actin (Sigma cat. no. A5441, 1:10,000). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 45 min at room temperature. Proteins were visualized using Novex ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) and a ChemiDoc XRS + imaging system (Bio-Rad). Densitometry was performed using the Chemidoc XRS + System image analysis software (Bio-Rad).