Sample: Lane 1: Mouse Cerebellum tissue lysates Lane 2: Rat Cerebellum tissue lysates Lane 3: Human Raji cell lysates Lane 4: Human HeLa cell lysates Lane 5: Human SH-SY5Y cell lysates Lane 6: Human U251 cell lysates Lane 7: Human 293T cell lysates Primary: Anti- MAPK4 (bs-4131R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 66 kDa Observed band size: 60 kDa

Paraformaldehyde-fixed, paraffin embedded (human cervical carcinoma); 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 (MAPK4) Polyclonal Antibody, Unconjugated (bs-4131R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat cerebellum); 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 (MAPK4) Polyclonal Antibody, Unconjugated (bs-4131R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat kidney tissue); 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 (ERK4) Polyclonal Antibody, Unconjugated (bs-4131R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat brain tissue); 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 (ERK4) Polyclonal Antibody, Unconjugated (bs-4131R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Tissue/cell: mouse brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-ERK4 Polyclonal Antibody, Unconjugated(bs-4131R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-ERK4 Polyclonal Antibody, Unconjugated(bs-4131R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum); 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 (MAPK4) Polyclonal Antibody, Unconjugated (bs-4131R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

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 (MAPK4) Polyclonal Antibody, Unconjugated (bs-4131R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse 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 (MAPK4) Polyclonal Antibody, Unconjugated (bs-4131R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control（black line）:Hela. Primary Antibody (green line): Rabbit Anti-MAPK4 antibody (bs-4131R) Dilution:1ug/Test; Secondary Antibody（white blue line）: Goat anti-rabbit IgG-AF488 Dilution: 0.5ug/Test. Isotype control（orange line）: Normal Rabbit IgG Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃, 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.