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Fig. 1. Expression of main hnRNP A/B proteins at different cell cycle stages. (A) Colo16 and HaCaT cells were synchronized by double thymidine block and DNA contents were analysed by flow cytometry to determine when S, G2, M and G1 phases occur. (B) Specificities of antibodies against hnRNPs A1, A2, A3 and B1 were confirmed by western blotting. Total proteins isolated from HaCaT cells were separated on SDS/12% polyacrylamide gels, blotted onto PVDF membrane, probed with antibodies against hnRNP A1, A2, A3, or B1 and detected with ECL-Plus. (C) The synchronized cells were collected at different times after the release of second thymidine block with the sampling times indicated at the top of lanes. For Colo16 cells, S, G2, M and G1 phase started at 0, 7, 8 and 10 hours after synchronization, respectively, whereas HaCaT cells entered G2, M and G1 phases 1 hour later. Levels of hnRNP A/B proteins were determined by western blotting following the same protocol as above. The membranes were then stripped, and re-probed with a mouse anti-GAPDH antibody to ensure a same loading ratio of samples. Band intensities were measured by densitometry to assess the relative abundance of the target protein against GAPDH, which was indicated under the lanes. The experiments were performed in duplicate.
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