phospho-JNK1 + 2 + 3 (Thr183+Tyr185) Polyclonal Antibody

Image kindly provided by Dr. Magdalena Krol. Control tumor cells, tumor cells grown in macrophage-conditioned medium, tumor cells sorted from co-culture with macrophages, and macrophages from monocultures and sorted from co-culture with tumor cells were analyzed. Total protein concentrations in lysates were determined using a Bio-Rad protein assay. Proteins (50 mg) were resolved using SDS-PAGE and transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat dry milk in TBS buffer containing 0.5% Tween 20. The membranes were then incubated overnight with the primary Rabbit Anti-JNK1+2+3 (THR183+TYR185) at 1:100 dilution. Subsequently, the membranes were washed three times in TBS containing 0.5% Tween 20 and incubated for 1 h at room temperature with secondary antibodies conjugated with the appropriate infrared (IR) fluorophore IRDyeH 800 CW or IRDyeH 680 RD at a dilution of 1:5000.

Mouse splenocytes probed with Rabbit Anti-JNK1\/2\/3 (Thr183+Tyr185) Polyclonal Antibody, Unconjugated (bs-1640R) (green) at 1:50 for 30 minutes followed by a FITC conjugated secondary antibody compared to unstained cells (blue), secondary only(light blue), and isotype control(orange).

Paraformaldehyde-fixed, paraffin embedded Human brain glioma; 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:400 overnight at 4\u00b0C, 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:400 overnight at 4\u00b0C, DAB staining.

Lane 1: Rat Cerebrum lysates; Lane 2: Mouse Cerebrum lysates; Lane 3: Rat Cerebellum lysates; Lane 4: Mouse Cerebellum lysates; Lane 5: Rat Heart lysates; Lane 6: Mouse Heart lysates; Lane 7: Mouse Kidney lysates probed with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:1000 dilution and 4˚C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.

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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.

Paraformaldehyde-fixed, paraffin embedded Mouse heart; 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.

Paraformaldehyde-fixed, paraffin embedded Rat kidney; 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.

Paraformaldehyde-fixed, paraffin embedded Human colon cancer; 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.

Lane 1: Rat Cerebrum lysates; Lane 2: Mouse Cerebrum lysates; Lane 3: Rat Cerebellum lysates; Lane 4: Mouse Cerebellum lysates; Lane 5: Rat Heart lysates; Lane 6: Mouse Heart lysates; Lane 7: Mouse Kidney lysates probed with JNK1+2+3 Polyclonal Antibody, Unconjugated (bs-1640R) at 1:1000 dilution and 4˚C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.

Jurkat cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20\u2103, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with JNK1+2+3 (T183+Y185) Antibody(bs-1640R)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 incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).

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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.

Paraformaldehyde-fixed, paraffin embedded Human 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.

Paraformaldehyde-fixed, paraffin embedded Human gastric 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.

Paraformaldehyde-fixed, paraffin embedded Mouse kidney; 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\u00b0C for 30min; Antibody incubation with JNK1+2+3 (T183+Y185) Polyclonal Antibody, Unconjugated (bs-1640R) at 1:200 overnight at 4\u00b0C, DAB staining.