Blank control (blue line): A431 cells(fixed with 70% ice-cold methanol overnight at 4℃). Primary Antibody (green line): Rabbit Anti-CHRM4/PE-CY7 Conjugated antibody (PC11998-PE-CY7),Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG-PE-CY7 .

Blank control: K562. Primary Antibody (green line): Rabbit Anti-CHRM4 antibody (PC11998) Dilution: 2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-AF488 Dilution: 1μg /test. Protocol The cells were incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.

Black line : Positive blank control A431); Negative blank control (Huvec） Green line : Primary Antibody (Rabbit Anti-CHRM4 antibody (PC11998) ) Orange line：Isotype Control Antibody (Rabbit IgG) . Blue line : Secondary Antibody (Goat anti-rabbit IgG-AF488) A431（Positive）and HUVEC Negative control）cells (black) were incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with CHRM4 Antibody(PC11998)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).