Phospho-NDRG1 (T346) Recombinant Rabbit Monoclonal Antibody [PSH07-17]

☑ Cell treatment (CT)

Western blot analysis of Phospho-NDRG1 (T346) on different lysates with Rabbit anti-Phospho-NDRG1 (T346) antibody (HA722805) at 1/2,000 dilution.

Lane 1: HT-29 cell lysate (25 µg/Lane)
Lane 2: HT-29 treated with 100nM Calyculin A for 30 minutes cell lysate (25 µg/Lane)
Lane 3: HeLa cell lysate (25 µg/Lane)
Lane 4: HeLa serum starved for 4 hours then add 100nM insulin for 5 minutes cell lysate (25 µg/Lane)
Lane 5: C2C12 cell lysate (25 µg/Lane)
Lane 6: C2C12 treated with 100nM insulin for 15 minutes cell lysate (25 µg/Lane)
Lane 7: NIH/3T3 cell lysate (25 µg/Lane)
Lane 8: NIH/3T3 serum starved for 24 hours then add 100nM Calyculin A for 30 minutes cell lysate (25 µg/Lane)

Predicted band size: 43 kDa
Observed band size: 50 kDa

Exposure time: 20 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 (HA722805) 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.

☑ Cell treatment (CT)

Immunocytochemistry analysis of 293T cells treated with 100nM Calyculin A for 15 minutes labeling Phospho-NDRG1 (T346) with Rabbit anti-Phospho-NDRG1 (T346) antibody (HA722805) 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-NDRG1 (T346) antibody (HA722805) 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.

☑ Cell treatment (CT)

Western blot analysis of Phospho-NDRG1 (T346) on different lysates with Rabbit anti-Phospho-NDRG1 (T346) antibody (HA722805) at 1/2,000 dilution.

Lane 1: 293T cell lysate
Lane 2: 293T treated with 100nM Calyculin A for 15 minutes cell lysate
Lane 3: 293T treated with 100nM Calyculin A for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 43 kDa
Observed band size: 50 kDa

Exposure time: 10 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 (HA722805) 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-NDRG1 (T346) on C6 cell lysates with Rabbit anti-Phospho-NDRG1 (T346) antibody (HA722805) at 1/2,000 dilution.

Lysates/proteins at 20 µg/Lane.

Predicted band size: 43 kDa
Observed band size: 50 kDa

Exposure time: 30 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 (HA722805) 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.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-Phospho-NDRG1 (T346) antibody (HA722805) at 1/2,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722805) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-Phospho-NDRG1 (T346) antibody (HA722805) at 1/2,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722805) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Flow cytometric analysis of 293T cells labeling Phospho-NDRG1 (T346).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722805, 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).