Fig. 6.
Osmotic stress decreases RNA binding to hDlg and PSF. (A) Endogenous PSF or hDlg were immunoprecipitated, with anti-PSF or anti-hDlg antibody, from HEK293 cells, alone or treated with 0.5 M sorbitol (15 minutes); IgG immunoprecipitation was used as negative control. RNA in the pellets was purified by a differential display-based method using set 1 or set 2 of random primers and resolved on a 6% denaturing polyacrylamide gel. Bands of interest were excised, re-amplified and cloned for identification (bands are indicated as 1 to 13; unidentified bands are labelled n.d.); band identity and GenBank accession number are shown (bottom). (B) Extracts from were prepared as in A, hDlg or PSF immunoprecipitated and the levels of bound RNA identified in A were determined using specific primers for each RNA by real-time PCR. The RNAs identified were also amplified by RT-PCR using specific primers and resolved in an agarose gel. (C) WT, p38γ171A/171A or p38γ−/− MEF extracts were prepared as in A, hDlg was immunoprecipitated and the levels of bound ODC1 RNA determined using specific primers by real-time PCR.