Proteases and phosphatase inhibitors

Whole cell extracts were prepared from ND-MK and MF-MK, MNC, and normal MSC cultures, in NP40 buffer plus proteases and phosphatase inhibitors (Roche). Total proteins (30–50 μg) were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Transferred proteins were detected first with primary antibodies pSMAD2 (3108), SMAD2/SMAD3 (3102), pJNK (4668), JNK (9258), pSTAT3 (9145), STAT3 (9139), p53 (9282), GATA2 (4595), p21 (2947; Cell Signaling); GAPDH (CB1001, Calbiochem); p57^Kip2 (ab119989, Abcam); and then with appropriate horseradish peroxidase–coupled secondary antibodies (Calbiochem). Immune complexes were detected with an enhanced chemiluminescence kit (Amersham) or LUM-LIGHT^Plus (Roche).

Cell lysates were prepared from infected or uninfected cells at indicated time points post infection (p.i.) with the Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime) supplemented with proteases and phosphatase inhibitors (Roche, Basel, Switzerland). Protein samples were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, United States) and probed with primary antibodies, which were further bound to HRP-conjugated secondary antibodies after washes. Finally, the PVDF membrane was immersed by Immun-Star™ HRP Substrate (Bio-Rad, Hercules, CA, United States) and images were captured using a ChemiDoc™ XRS + imaging system (Bio-Rad). Digital signal acquisition and analysis were conducted using the Quantity One program, version 4.6 (Bio-Rad).