Fig. 1.

Association of signal transducer and activator of transcription 5a (STAT5a) with endoplasmic reticulum (ER) sheets and the ER-resident GTPase atlastin-3 (ATL3). A: human pulmonary arterial endothelial cell (HPAEC) cultures in 6-well plates were fixed with paraformaldehyde and then permeabilized with buffers containing either 0.1% Triton X-100 or 50 μg/ml digitonin followed by immunofluorescence imaging as indicated. Reticulon-4 (RTN4) is an ER structural protein. Arrows in the top row point to the Golgi apparatus; arrowheads in the bottom two rows highlight regions of ER sheets. Scale bars = 10 μm. B: equal aliquots of endothelial cell (EA.hy926 cells) extracts were subjected to magnetic-bead immunopanning (IPN) using anti-STAT5a pAb (in triplicate) and anti-STAT3 pAb as a negative control. The analysis included a peptide competition cross-IPN using excess of the relevant STAT5a peptide or an irrelevant STAT3 peptide. The immunoisolates were sequentially Western blotted as indicated. C: equal aliquots of HPAEC extracts were subjected to magnetic-bead IPN using anti-STAT5a or anti-STAT3 pAb. The immunoisolates were sequentially Western blotted as indicated.