Sc 166355

SiHa and C33A cells were lysed in a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.5% NP-40) containing protease and phosphatase inhibitors with a Mini tablet (Thermo Fisher, A32961) added to 10 mL of the lysis buffer. The expression of FBXO22, p57^Kip2, and GAPDH was examined by Western blotting analysis as previously described [31 (link)]. The following primary antibodies were used for Western blotting analysis: mouse anti-FBXO22 (1:500, sc-100736, Santa Cruz), rabbit anti-p57 (1:1000, sc-1040, Santa Cruz), mouse anti-Ub (1:500, sc-166355, Santa Cruz), mouse anti-GAPDH (1:2500, 80602-840, Calbiochem) Image J software was used for the measurement of band density and relative expression of a protein the ratio of its band density normalized by that of its corresponding GAPDH protein band.

MaxiSorp™ plates were coated at 4 °C overnight with mouse anti-CD81 antibody (1:100, sc-23962, Santa-Cruz Biotechnology Inc., Santa Cruz, CA, USA). Then, saturation was processed with 100 µL/well of 1X assay diluent for 1 h under agitation. CCS of HEK293 stably expressing ZIKV-NS1^FLAG-tag was incubated for 2 h at room temperature. After a washing step, a mouse anti-FLAG antibody (1:100, sc-166355, Santa-Cruz Biotechnology Inc., Santa Cruz, CA, USA) was incubated for 1 h at room temperature under agitation. Finally, a goat anti-mouse-HRP antibody (ab6789, Abcam, Cambridge, UK) was added for 30 min at room temperature. Between each incubation step, wells were washed with PBS-Tween 0.05% three times. HRP activity was revealed using 50 μL of 3,3′, 5,5′-tetramethylbenzidine (TMB solution, 00-4201-56, eBioscence, Inc., San Diego, CA USA, Invitrogen) as HRP substrate, and stopped with 50 µL of HCL 0.2 N. Absorbance was read at 450 nm, with a reference at 570 nm, using a Fluostar Omega microplate reader (BMG LabTech).

Gels for SDS-PAGE or Phos-tag SDS-PAGE were prepared according to the manufacturer’s instructions (NARD Institute). Separated proteins were transferred onto a PVDF membrane and then were identified by immunoblot analysis with the appropriate primary antibodies at a dilution of 1:1000 (or as otherwise stated below). Antibody to Mob1 phosphorylated at Thr35 (1:2000 dilution; 8699), anti-Mob1 (1:2000 dilution; 13730), anti-Mst1 (1:2000 dilution; 3682), anti-Mst2 (1:2000 dilution; 3952), anti-GAPDH (1:5000 dilution; 5174), antibody to phosphorylated p38 (1:3000 dilution; 9211), antibody to phosphorylated Jnk (1:3000 dilution; 4668), and anti-β-actin (1:5000 dilution; 8457) were from Cell Signaling Technology. Anti-Rac1 (66122-1-Ig), anti-HA Rabbit (51064-2-AP), and anti-Flag Rabbit (1:3000 dilution; 20543-1-AP) were from Proteintech. Anti-gamma Tubulin (1:5000 dilution; ab11316) was from Abcam and anti-Flag Mouse (1:5000 dilution; sc-166355) was from Santa Cruz Biotechnology. The protein bands were visualized with a SuperSignal West Pico Kit according to the manufacturer’s instructions (Thermo Fisher Scientific Pierce).