Anti-p62/SQSTM1 Rabbit Monoclonal Antibody

Western blot analysis of P62/SQSTM1 using anti-P62/SQSTM1 antibody (M00300).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Hela- WT whole cell lysates,
Lane 2: human Hela-SQSTM1 KO whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-P62/SQSTM1 antigen affinity purified monoclonal antibody (M00300) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for P62/SQSTM1 at approximately 62 kDa. The expected band size for P62/SQSTM1 is at 48 kDa.

Western blot analysis of p62/SQSTM1 using anti-p62/SQSTM1 antibody (M00300).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human HepG2 whole cell lysates,
Lane 2: human A549 whole cell lysates,
Lane 3: human RT4 whole cell lysates,
Lane 4: human Hela whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat PC-12 whole cell lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse NIH/3T3 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-p62/SQSTM1 antigen affinity purified monoclonal antibody (Catalog # M00300) at 1:5000 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:1000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for p62/SQSTM1 at approximately 62 kDa. The expected band size for p62/SQSTM1 is at 48 kDa.

IHC analysis of p62/SQSTM1 using anti-p62/SQSTM1 antibody (M00300).
p62/SQSTM1 was detected in a paraffin-embedded section of human colorectal adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-p62/SQSTM1 Antibody (M00300) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of p62/SQSTM1 using anti-p62/SQSTM1 antibody (M00300).
p62/SQSTM1 was detected in a paraffin-embedded section of human spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-p62/SQSTM1 Antibody (M00300) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Immunofluorescent analysis of Hela cells, using p62/SQSTM1 Antibody.

IHC analysis of p62/SQSTM1 using anti-p62/SQSTM1 antibody (M00300).
p62/SQSTM1 was detected in a paraffin-embedded section of human cervix squamous cell carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-p62/SQSTM1 Antibody (M00300) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Immunofluorescent analysis using the Antibody at 1:50 dilution.

The depletion of β-catenin triggered by PPI is mediated through the process of autophagy. ( A , B ) The effect of different concentrations of PPI on the expression levels of β-catenin protein in HO-8910PM cells following a 6-h treatment was assessed using a Western blot assay, and the resulting data were further subjected to quantitative analysis. ( C ) qRT-PCR analysis of β-catenin mRNA levels in PPI-treated cells. ( D ) Assessment of the impact of PPI on β-catenin protein degradation: HO-8910PM cells were exposed to PPI (1 μM) either alone or in combination with either MG132 (20 μM), which is a proteasome inhibitor, or BAF (200 nM), which is an autophagy inhibitor, for 6 h. Subsequently, the cells were subjected to CHX (100 µg/mL) treatment for the indicated durations. ( E ) The quantification results of ( D ). ( F , G ) The effect of different concentrations of PPI on the expression levels of p62 protein in HO-8910PM cells following a 6-h treatment was assessed by WB, and the resulting data were subjected to quantitative analysis. ( H – J ) The effect of different concentrations of PPI on the expression levels of LC3 I and LC3 II proteins in HO-8910PM cells following a 6-h treatment was assessed by WB, and the resulting data were subjected to quantitative analysis. Data from six independent experiments are presented as mean ± S.D. *: p < 0.05; **: p < 0.01; ***: p < 0.001 vs. control; ###: p < 0.001 vs. 1 μM PPI.
Index in PubMed under a CC BY license. PMID: 40429774