p62/KEAP1/NRF2 Pathway Antibody Sampler Kit

Western blot analysis of NQO1 on different lysates with Rabbit anti-NQO1 antibody (ET1702-50) at 1/2,000 dilution.

Lane 1: HCT 116-si NT cell lysate
Lane 2: HCT 116-si NQO1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 31 kDa
Observed band size: 31 kDa

Exposure time: 16 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 (ET1702-50) 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 human breast cancer tissue with Rabbit anti-NQO1 antibody (ET1702-50) at 1/1,000 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 (ET1702-50) at 1/1,000 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.

Application: IHC-Fr

Species: Mouse

Site: Cerebral cortex

Sample: Frozen section

Antibody concentration: 1/500

Antigen retrieval: The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven.

Application: IF-tissue

Species: Mouse

Site: Liver

Sample: Paraffin-embedded section

Antibody concentration: 1/500

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-SQSTM1 / p62 antibody (HA721171) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA721171) at 1/1,000 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.

Western blot analysis of Phospho-SQSTM1 / p62 (S349) on different lysates with Rabbit anti-Phospho-SQSTM1 / p62 (S349) antibody (HA723050) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 2μM MG-132 for 18 hours cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 10μM MG-132 for 8 hours cell lysate
Lysates/proteins at 20 µg/Lane.

Predicted band size: 48 kDa
Observed band size: 62 kDa

Exposure time: 11 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 (HA723050) 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.

Immunocytochemistry analysis of HeLa cells treated with 2μM MG-132 for 18 hours labeling Phospho-SQSTM1 / p62 (S349) with Rabbit anti-Phospho-SQSTM1 / p62 (S349) antibody (HA723050) at 1/5,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-SQSTM1 / p62 (S349) antibody (HA723050) at 1/5,000 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 (HA601187, 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.

Western blot analysis of MAP1LC3A on different lysates with Rabbit anti-MAP1LC3A antibody (ET1609-26) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 50μM Chloroquine for 18 hours cell lysate
Lane 3: C2C12 cell lysate
Lane 4: C2C12 treated with 50μM Chloroquine for 18 hours cell lysate
Lane 5: C6 cell lysate
Lane 6: C6 treated with 50μM Chloroquine for 18 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 14 kDa
Observed band size: 14/16 kDa

Exposure time: 3 minutes; 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 (ET1609-26) 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 C6 cells labeling MAP1LC3A with Rabbit anti-MAP1LC3A antibody (ET1609-26) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-MAP1LC3A antibody (ET1609-26) 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 (HA601187, 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.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-Keap1 antibody (HA721525) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA721525) at 1/1,000 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.

Western blot analysis of Nrf2 on different lysates with Rabbit anti-Nrf2 antibody (HA723302) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 2μM MG-132 for 18 hours cell lysate
Lane 3: C6 cell lysate
Lane 4: C6 treated with 25μM MG-132 for 4 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 68 kDa
Observed band size: 100 kDa

Exposure time: 2 minutes; 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 (HA723302) 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.

Immunocytochemistry analysis of MEF cells untreated / treated with 2μM MG-132 for 18 hours labeling Nrf2 with Rabbit anti-Nrf2 antibody (HA723302) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-Nrf2 antibody (HA723302) 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 (HA601187, 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.

Chromatin immunoprecipitations were performed with cross-linked chromatin from MEF cells treated with 10μM MG-132 for 8 hours with Nrf2 (HA723302) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.