R pe conjugated secondary antibody

Cultured and pre-treated PM_ϕ (n ≥ 2 ^∗ 10⁶ cells per treatment group) were fixed and permeabilized with pre-chilled methanol–acetone (ratio 1:1). Non-specific antibody binds were blocked with 1% BSA in PBS for 1 h. PM_ϕ 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, and the cytoplasmic membranes were stained with cholera toxin B (CTxB) (C34775, Thermo Fisher Scientific) for 30 min at 4°C. DAPI nuclei staining were done and followed by imaging and assessment of the ratios of nucleic and cytoplasmic GRK5 expressions with ImageJ (n = 12–15 cells per four mice).

Formalin (4%) was used to fix cultured and treated H9c2 cells (n ≥ 2 ∗ 10⁶ cells per treatment group). Triton x (0.1%)(VICMED; 9005–64-5) was used to permeate cells for 15 min. Binding sites of non-specific antibodies were blocked for 1 h in PBS with 1% BSA. H9c2 cells were then treated and incubated overnight at a constant temperature of 4 °C with ADAM17 primary antibody (Abcam; ab2051), then rinsed with PBS, and probed with R-PE-conjugated secondary antibody (Proteintech; SA00008-2) for 1 h under room temperature. Subsequently, the cells were washed with PBS and conditioned with 0.5% BSA in Hanks' balanced salt solution. The cytoplasmic membranes of cells were then stained for 30 min at 4 °C with WGA (Thermo Fisher Scientific; W11261). DAPI (Beyotime; C1005) nuclei staining was performed, followed by imaging and assessment of membrane, cytoplasmic, and nucleic expressions ratios of ADAM17 with ImageJ (n = 25–35 cells per disk).