Phospho-Tau (Ser214/T217) Signaling Antibody Sampler Kit

Western blot analysis of JNK1+JNK2+JNK3 on different lysates with Rabbit anti-JNK1+JNK2+JNK3 antibody (ET1601-28) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HEK-293 cell lysate
Lane 3: Jurkat cell lysate
Lane 4: MCF7 cell lysate
Lane 5: NIH/3T3 cell lysate
Lane 6: PC-12 cell lysate
Lane 7: C6 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 48/53 kDa
Observed band size: 48/53 kDa

Exposure time: 35 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-28) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of p38 alpha on different lysates with Rabbit anti-p38 alpha antibody (ET1702-65) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: Jurkat cell lysate
Lane 3: HEK-293 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: Neuro-2a cell lysate
Lane 6: RAW264.7 cell lysate
Lane 7: PC-12 cell lysate
Lane 8: C6 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 41 kDa
Observed band size: 38 kDa

Exposure time: 3 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1702-65) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of Phospho-p38 alpha (T180 + Y182) on different lysates with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) at 1/2,000 dilution and p38 alpha antibody (ET1702-65) at 1/1,000 dilution.

Lane 1: Jurkat cell lysate (20 µg/Lane)
Lane 2: Jurkat treated with UV for 1 hour cell lysate (20 µg/Lane)
Lane 3: NIH/3T3 cell lysate (20 µg/Lane)
Lane 4: NIH/3T3 treated with 25μg/mL Anisomycin for 30 minutes cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 25μg/mL Anisomycin for 30 minutes cell lysate (20 µg/Lane)

Predicted band size: 41 kDa
Observed band size: 38 kDa
Exposure time: 3 minutes; ECL: K1802;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722669) at 1/2,000 dilution and p38 alpha antibody (ET1702-65) at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of Phospho-JNK1/2/3 (T183 + T183 + T221) on different lysates with Rabbit anti-Phospho-JNK1/2/3 (T183 + T183 + T221) antibody (ET1609-42) at 1/2,000 dilution.

Lane 1: C6 cell lysate
Lane 2: C6 treated with 25ug/mL Anisomycin for 30 minutes whole cell lysate.

Lysates/proteins at 20 µg/Lane.
Predicted band size: 48/53 kDa
Observed band size: 48/53 kDa
Exposure time: 2 minutes 6 seconds;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-42) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of Phospho-p70 S6 Kinase (T389) on different lysates with Rabbit anti-Phospho-p70 S6 Kinase (T389) antibody (HA721803) at 1/1,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: MCF7 starved for 4 hours then treated with 200ng/mL EGF for 15 minutes cell lysate
Lane 3: C6 cell lysate
Lane 4: C6 treated with 100nM Calyculin A for 30 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 59 kDa
Observed band size: 70/85 kDa

Exposure time: 46 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721803) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of Jurkat cells treated with UV for 1 hour labeling Phospho-p38 alpha (T180 + Y182) with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) at 1/50 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunocytochemistry analysis of NIH/3T3 cells treated with 25μg/mL Anisomycin for 30 minutes labeling Phospho-p38 alpha (T180 + Y182) with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-p38 alpha (T180 + Y182) antibody (HA722669) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of Jurkat cells untreated (left) or treated (right) with UV for 1 hour labeling Phospho-p38 alpha (T180 + Y182).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722669, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Immunofluorescence analysis of frozen mouse hippocampus tissue labeling Phospho-JNK1/2/3 (T183 + T183 + T221) with Rabbit anti-Phospho-JNK1/2/3 (T183 + T183 + T221) antibody (ET1609-42).

The tissues were blocked in 3% BSA for 30 minutes at room temperature, washed with PBS, and then probed with the primary antibody (ET1609-42, green) at 1/100 dilution overnight at 4℃, washed with PBS. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) was used as the secondary antibody at 1/200 dilution. Nuclei were counterstained with DAPI (blue). Image acquisition was performed with KFBIO KF-FL-400 Scanner.

Immunofluorescence analysis of frozen mouse brain tissue with Rabbit anti-Tau antibody (ET1612-44) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1612-44, green) at 1/500 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Immunofluorescence analysis of frozen rat brain tissue with Rabbit anti-Tau antibody (ET1612-44) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1612-44, green) at 1/500 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

p70 S6 Kinase was immunoprecipitated from 0.2 mg C6 cell lysate with HA722520 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722520 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: C6 cell lysate (input)
Lane 2: HA722520 IP in C6 cell lysate
Lane 3: Rabbit IgG instead of HA722520 in C6 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 20 seconds; ECL: K1801

Immunocytochemistry analysis of MCF7 cells treated with or without 20% FBS overnight then add 100nM Calyculin A for 30 minutes labeling Phospho-p70 S6 Kinase (T389) with Rabbit anti-Phospho-p70 S6 Kinase (T389) antibody (HA721803) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-p70 S6 Kinase (T389) antibody (HA721803) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.