Formaldehyde-fixed and paraffin-embedded clinical HCC samples were examined for P-JNK (Cell Signaling Technology, Beverly, MA) and RACK1 (BD Biosciences, San Jose, CA) staining on tissue microarray slides (US Biomax, Inc., Rockville, MD; see detailed clinicopathological features in Supporting Table 2). Patients’ consent and approval by the local ethics committee were obtained for the use of the clinical materials in research. Male athymic BALB/c nude mice were purchased from the Institutes

of Experimental Animals, Academy of Chinese Medical Sciences (Beijing, China), and maintained under specific pathogen-free conditions. All experiments were performed in accord with institutional guidelines Selleckchem Vemurafenib for animal care. Six- to eight-week-old nude mice were subcutaneously inoculated

with human HCC cells (1 × 106/0.2 mL of phosphate-buffered saline; n = 10). Lengths and widths of tumors were measured with a caliper at the indicated time points. A full description of additional Materials and Methods are given in the Supporting Materials. RACK1 was shown to bind MKK7 in the yeast two-hybrid system (Supporting Table Sirolimus 1). The interaction of RACK1 with MKK7 was confirmed by coimmunoprecipitation (Co-IP) analysis: Green fluorescent protein (GFP)-MKK7 coprecipitated with coexpressed FLAG-RACK1, and FLAG-RACK1 coprecipitated with selleck screening library coexpressed GFP-MKK7 in human embryonic kidney 293T cells (Fig. 1A,B). To test whether RACK1 interacts directly with MKK7, we performed in vitro glutathione S-transferase (GST) pull-down assays. As expected, substantial FLAG-RACK1 and endogenous RACK1 in lysates of 293T cells was precipitated specifically by GST-MKK7, but not by GST alone (Fig. 1C). Moreover, in vitro translated FLAG-RACK1 was also precipitated specifically

by GST-MKK7, but not by GST alone (Fig. 1D). The possible physiological interaction of RACK1 with MKK7 in HCC cells was then analyzed by immunoprecipitating endogenous proteins. MKK7 was present in immunoprecipitates obtained from lysates of HepG2 human HCC cells with an antibody (Ab) against RACK1, whereas no MKK7 coprecipitated when we used a control Ab (healthy rabbit immunoglobulin G; IgG) (Fig. 1E). Moreover, endogenous RACK1 in HepG2 cells coprecipitated with endogenous MKK7 (Fig. 1F). Collectively, our data suggest that RACK1 could engage in a direct interaction with MKK7 in human HCC cells.