Anti-KMT6/EZH2 Antibody Picoband®

Western blot analysis of KMT6/EZH2 using anti-KMT6/EZH2 antibody (A00050-1).
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 Jurkat whole cell lysates,
Lane 2: human K562 whole cell lysates,
Lane 3: human MCF-7 whole cell lysates,
Lane 4: rat spleen tissue lysates,
Lane 5: rat testis tissue lysates,
Lane 6: mouse spleen tissue lysates,
Lane 7: mouse testis tissue 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-KMT6/EZH2 antigen affinity purified polyclonal antibody (Catalog # A00050-1) 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for KMT6/EZH2 at approximately 98 kDa. The expected band size for KMT6/EZH2 is at 85 kDa.

Flow Cytometry analysis of K562 cells using anti-KMT6/EZH2 antibody (A00050-1).
Overlay histogram showing K562 cells stained with A00050-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-KMT6/EZH2 Antibody (A00050-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

HOTTIP regulates EZH2 to inhibit PTEN expression. Note: (a) catRAPID predicted binding region of HOTTIP and EZH2. (b) FISH analyzed the localization of EZH2 and HOTTIP in IR-K562 cells. (c) RIP-PCR were used to test the interaction between EZH2 and HOTTIP. ∗ p < 0.05 vs. IgG. (d) RT-qPCR and Western blot were used to measure EZH2 mRNA and protein level after knocking down HOTTIP. (e) Western blot analysis was used to measure EZH2 in BM-MNCs of CML patients. Right panel, bar chart of protein densitometric analysis. ∗∗∗ p < 0.001. RT-qPCR was used to detect EZH2 level in BM-MNCs of CML patients. Normalized to GAPDH. ∗∗∗ p < 0.001 vs. NC. Western blot analysis was used to measure EZH2 protein level in IR-K562 and K562 cells. Right panel, bar chart of protein densitometric analysis. ∗∗∗ p < 0.001; RT-qPCR was used to detect EZH2 mRNA level in CML cell lines (K562 and IR-K562). Normalized to GAPDH. ∗∗∗ P < 0.001 vs. NC. (f) RT-qPCR and Western blot were used to detect EZH2 and PTEN mRNA and protein levels after transfecting with specific sh-con or sh-EZH2. ∗∗∗ P < 0.001 vs. DMSO.
Index in PubMed under a CC BY license. PMID: 36117724

HOTTIP work through EZH2 to participate in drug resistance. Note: (a) IR-K562 cells were transfected with specific sh-HOTTIP or both sh-HOTTIP+sh-EZH2. The CCK-8 analysis was used to detect cell proliferation. (b) Cell apoptosis was detected by flow cytometry using Annexin V/APC/PI. The right panel shows the apoptosis rate from three independent experiments. (c) IR-K562 cells were prepared with different Imatinib concentrations for 48 h. The CCK-8 analysis was used to detect cell inhibition. (d) WB was used to detect protein levels.
Index in PubMed under a CC BY license. PMID: 36117724

Western blot analysis of EZH2 using anti-EZH2 antibody (A00050-1).
Electrophoresis was performed on a 8% 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 THP-1- WT whole cell lysates,
Lane 2: human THP-1-EZH2 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-EZH2 antigen affinity purified polyclonal antibody (A00050-1) 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 EZH2 at approximately 98 kDa. The expected band size for EZH2 is at 85 kDa.

Immunoprecipitating KMT6/EZH2 in MCF-7 whole cell lysate.
Western blot analysis of KMT6/EZH2 using anti-KMT6/EZH2 antibody (A00050-1).
Lane 1: MCF-7 whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-KMT6/EZH2 antibody in MCF-7 whole cell lysate;
Lane 3: anti-KMT6/EZH2 antibody (2μg) + MCF-7 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-KMT6/EZH2 antigen affinity purified polyclonal antibody (A00050-1) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1196-200). A specific band was detected for KMT6/EZH2 at approximately 98 kDa. The expected band size for KMT6/EZH2 is at 85 kDa.