Figure 4.

PDIA6 is an RNA binding protein. (A) Assessment of RNA-binding by OOPS. Left, schematic representation of the OOPS principle, including the Drosophila spike-in control. Right, western blot showing that PDIA6 is recovered in the OOPS eluate (output) upon UV-crosslinking. Crosslinked Drosophila extracts were added to all samples to estimate sample recovery, as monitored by Western blot against Stubarista (Sta). Vinculin (VCL) was used as negative control. (B) Assessment of RNA-binding by PNK assay. PDIA6 was immunoprecipitated from 2 mg of extract from SK-Mel-147 or Mel-ST cells, and the associated RNA digested and labelled with ³²P. The same membrane was first exposed to capture the ³²P-RNA signal and then incubated with anti-PDIA6 antibodies for western blot (WB). The asterisk denotes a non-specific band. Quantification of 3 independent experiments is shown on the right. (C) PDIA6 binds RNA in a panel of melanoma cell lines. RNA-binding was assessed by OOPS as in (A). Only crosslinked samples are shown. Quantification of the output signal corrected for input and spike in, and normalized to SK-Mel-147 is shown on the right. Significance was assessed by Student's t-test (n = 3–4).