PAR1 regulates the expression of melanoma cell adhesion molecule MUC18 in metastatic melanoma. A, PAR1 silencing (shPAR1) using lentiviral shRNA results in down-regulation of PAR1 expression in A375SM and C8161-c9 cells as compared with control cells transduced with the nontargeting shRNA (shNT). Cell lysates were immunoprecipitated using anti-PAR1 antibody, and PAR1 expression was further detected by Western blot. The IgG band served as an indicator of equal loading. B, Western blot analysis demonstrates that PAR1 silencing results in down-regulation of MUC18 expression in A375SM and C8161-c9 cells as compared with control cells transduced with the nontargeting shRNA. C, stable re-expression of PAR1 (shPAR1/PAR1) in A375SM or C8161-c9 PAR1-silenced cells using lentiviral transduction as demonstrated by immunoprecipitation with anti-PAR1 antibody followed by Western blot. Note an increase in PAR1 levels in shPAR/PAR1 cells as compared with shPAR1 cells transduced with the empty expression vector (shPAR1/ev). The IgG band served as an indicator of equal loading. Please note that the loading order in C8161-c9 cells (right panel) is different from A375SM cells (left panel). D, re-expression of PAR1 (shPAR1/PAR1) in A375SM or C811-c9 PAR1-silenced cells rescues MUC18 expression as compared with PAR1-silenced cells transduced with the empty expression vector (shPAR1/ev).