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Sample: Brain (Rat) Lysate at 40 ug Primary: Anti-Phospho-RhoA(Ser188) (bs-5330R) at 1/300 dilution Secondary: HRP conjugated Goat-Anti-rabbit IgG (bs-0295G-HRP) at 1/5000 dilution Predicted band size: 21 kD Observed band size: 21 kD

Paraformaldehyde-fixed, paraffin embedded (Rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Phospho-RhoA(Ser188)) Polyclonal Antibody, Unconjugated (bs-5330R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Molt-4 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 0.1% PBST for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Phospho-RhoA Antibody(bs-5330R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

Blank control (Black line): Molt4 (Black). Primary Antibody (green line):Rabbit Anti-Phospho-RhoA (Ser188) antibody (bs- 5330R) Dilution: 3μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647 Dilution: 3μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with PBST for 20 min at room temperature. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min 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.