Immunofluorescence staining
Cultured and pre-treated PMɸ were fixed and
permeabilized with pre-chilled methanol-acetone of ratio 1:1.
Non-specific antibody binds were blocked with 1% BSA in PBS for 1 h.
PMɸ were then incubated with GRK5 primary antibody
(ab64943; Abcam) at 4°C overnight, washed with PBS, and probed with
R-PE-conjugated secondary antibody (SA00008-2; Proteintech) at room
temperature for 1 h. Next, cells were washed with PBS, conditioned with
0.5% BSA in Hanks’ balanced salt solution (HBSS), and the cytoplasmic
membranes were stained with cholera toxin B (CTxB) (C34775, ThermoFisher
Scientific) for 30 min at 4°C. DAPI nuclei stainings were done and
followed by imagings. ImageJ was used to measure cytoplasmic and nuclei
regions of interest. The measured values were corrected for background
signal, and nucleic and cytoplasmic GRK5 expression ratios were
calculated.