*2.6. Immunofluorescence Assays*

Cells seeded on coverslips or chamber slides and used for experiments were washed 1x with PBS and were fixed with 4% paraformaldehyde in TBS for 30 min at room temperature. Following this, cells were washed with filtered 1X TBS and permeabilized (for pY-STAT staining, fixation was for 10 min at −20 ◦C with 100% ice-cold methanol and drying cells completely, followed by a further 3 washes with TBS; for all other samples, fixed cells were permeabilized via addition of 0.1% Triton X-100 to the blocking bu ffer, with permeabilization occurring during the blocking step). Blocking of cells was for 30 min at RT with filtered 5% normal goa<sup>t</sup> serum (NGS) in TBS. Cells were then stained for 1 h at RT or overnight at 4 ◦C with primary antibody in 5% NGS/TBS. Subsequently, cells were washed 3x with TBS and probed for 1 h at RT with secondary antibody plus 1:10,000 DAPI (4',6-diamidino-2-phenylindole dihydrochloride) in 5% NGS/TBS. Finally, cells were washed 3x with TBS; briefly washed 1x with deionized water (dH2O) to remove excess salt; and carefully dried on the back of the coverslip, removing excess water. Coverslips were mounted onto slides using ProLong Gold mounting media and dried at least overnight at RT. Confocal immunofluorescence images were acquired on a Nikon Eclipse Ti microscope and analyzed using the NIS-Elements imaging system software (version 4.51) (Nikon Instruments, Tokyo, Japan). For some images, the red channel was modified equally via a saved look-up table (LUT) setting across all samples per experiment to aid visual clarity post-acquisition.
