Ribonucleotide Reductase Large Subunit M1 Predicts Poor Survival Due to Modulation of Proliferative and Invasive Ability of Gastric Cancer
Figure 1
Determining RRM1 expression and localization in GC cells and tissue samples.
A. Peptide blocking assay was conducted on the RRM1 antibodies for IHC staining, all antibodies were in 1∶1000 dilution and incubated with recombinant RRM1 peptide (1 µg/ml) at 4° overnight. Antibody #2 showed more specific and stronger signal than other antibodies (data not show), therefore we used it as the antibody for IHC staining. B. Nuclear fractionation was employed on AGS cells. AGS cells were starved for 96 hours and re-supplemented with normal growth medium for 12 hours. Then the cells were collected and fractionized for nuclear protein. Western blot was applied to reveal the sub-cellular localization of RRM1, GAPDH and Sp-1 were used as cytoplasmic and nuclear markers. C. Translocation of RRM1 from cytoplasm to nucleus was observed by Immuno-fluorescence cytochemistry. AGS cells were starved for 48 hours and re-supplemented with normal growth medium for 12 hours. Then the cells were fixed and incubated with primary and secondary antibodies. Translocation of RRM1 was observed under fluorescence microscopy. D. RRM1 was heterogeneously expressed among adjacent normal tissue and histological subtypes (JGCA classification V.2011) including papillary, tubular, mucinous and signet ring cell and undifferentiated adenocarcinoma.