Anti phospho samhd1 thr592

Proteins from cells were lysed with RIPA Lysis and Extraction Buffer (Thermos Fisher Scientific, Waltham, MA, USA). After being mixed with Laemmli buffer (BioRad, Hercules, CA, USA), protein samples were heated at 95 °C for 10 min. Protein samples were then separated by SDS-PAGE gel electrophoresis, and transferred onto PVDF membrane (Millipore, Billerica, MA, USA). After being probed with primary and secondary antibodies, protein bands in membranes were detected for chemiluminescence using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher, Rockford, lL, USA). The primary antibodies used were: anti-p21^Cip1 (#2947) and anti-phospho-SAMHD1 (Thr592) (#89930) (Cell Signaling Technologies, Danvers, MA, USA); anti-SAMHD1 (#12586–1-AP), anti-GAPDH (#60004–1-Ig) and anti-RRM2 (#11661–1-AP) (Proteintech Group, Inc. Rosemont, IL, USA); anti-p53 (#sc-126, Santa Crus, Dallas, TX, USA); anti-actin (#A5441, Sigma-Aldrich, St. Louis, MO, USA) and anti-tubulin (# N-356, Amersham, GE Healthcare, Pittsburgh, PA, USA). Western blot images were detected by the ChemiDoc XRS+ system (BioRad, Hercules, CA, USA), and image analysis was performed by using the Image Lab™ software (BioRad, Hercules, CA, USA).

HCV replicon cells were treated with 30 μM CDM-3008, iCDM-17, and iCDM-34 for 1 day. The cells were lyzed with 1 x SDS sample buffer containing PhosSTOP, Complete EDTA free, and 1 mM EDTA. After sonication for 30 s, the lysates were centrifuged at 12,000g for 10 min at 4 °C, and then the supernatants were collected. After reducing the 20 μg of supernatants with 50 mM DTT at 98 °C for 2 min, the samples were separated by SDS-PAGE. STAT1, STAT2, STAT3, pSTAT1, pSTAT2, and pSTAT3 were detected by Western blotting using anti-STAT1 (9175, 1/1000), STAT2 (72604, 1/1000), STAT3 (12640, 1/1000), pSTAT1 (Y701) (7649, 1/1000), pSTAT2 (Y690) (88410, 1/1000), and pSTAT3 (Y705) (9145, 1/1000) antibodies (Cell signaling technology, Massachusetts, USA).
HepG2-NTCP-C4 cells were treated with 30 μM iCDM-17 and iCDM-34 for 1 day. The cells were lyzed with 20 mM TrisHCl pH 7.5 containing 100 mM NaCl, 10 mM EDTA, 1% TritonX-100, 1% Sodium deoxycholate, PhosSTOP (Roche), Complete EDTA free. The lysates were sonicated 30 s and mixed for 1 h with rotation at 4 °C. The lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatants were analyzed by Western blotting using anti-phospho-SAMHD1 (Thr592) (Cell signaling technology, 89930, 1/1000), anti-SAMHD1 (Cell signaling technology, 49158, 1/1000), and β−actin (Santa Cruz, 1/1000).