Figure 2

RRM2-c2orf48 expression in NPC. (a) The expression level of RRM2-c2orf48 mRNA in nasopharyngeal carcinoma cell lines (CNE1 to HNE1) was higher than that in normal nasopharyngeal epithelium cells (N01–09) and in immortalized nasopharyngeal cell lines (NPEC1-2 Bmi-1) by real-time PCR. (b) The expression level of RRM2-c2orf48 mRNA in nasopharyngeal carcinoma specimens was higher than that in the control specimens by real-time PCR (P=0.03). (c) Amplification of RRM2-c2orf48 and wild-type RRM2, c2orf48 in C666-1 cDNA. Electrophoresis results of 1% agarose gel show a specific band with lengths of RRM2 (1170 bp, nm_001034), RRM2-c2orf48 (1125 bp) and c2orf48 (480 bp, nm_182626), respectively. (d) RRM2-c2orf48 (43 kd), RRM2 (45 kd) and c2orf48 (17 kd) expression was detected in HNE1 PMSCV or HNE1 PMSCV-flag-c2orf48/RRM2/c2orf48 cell lines using anti-flag antibodies by western blotting. (e) ELISA result of a new synthesized polyclonal antibody against the chimeric region of RRM2-c2orf48. (f) The band of RRM2-c2orf48 can be identified from anti-flag and synthesized antibody. (g) To confirm the specificity of synthesized antibody, immunofluorescence was used in NPC cell lines HNE1 and C666-1, expressing lower and higher RRM2-c2orf48, respectively. And RRM2-c2orf48 was located in cytoplasm. (h) RRM2-c2orf48 protein expression in most of the NPC cell lines was higher than that in NPEC1 Bmi-1