The protein level of KDM5A, p16, and Fbxo22 was detected using streptavidin peroxidase labeled by immunohistochemical peroxidase. Paraffin-embedded specimens of breast cancer tissues were serially sectioned (5 μm thickness) and immune-stained with primary antibodies of rabbit anti-KDM5A (1:1000, ab78322, Abcam, UK), rabbit anti-p16 (1:100, A0262, ABclonal), and rabbit anti-Fbxo22 (13,606–1-AP, Protein-tech, Wuhan, China) overnight at 4 °C. The samples were incubated at 37 °C for 20 min with biotin-labeled goat anti-rabbit secondary antibody (BA1003, Boster), followed by incubation of 50 μL of streptomyces anti-biotin–peroxidase solution for 10 min at ambient temperature. Color development was completed with 3,3′-diaminobenzidine and microscopic observation for the specimens was performed with IgG used as a NC. Protein-positive cells were identified by the brownish-yellow color of normal positive cells, and positive staining was statistically analyzed using ImageJ.
Ab78322
Manufactured by Abcam
Sourced in United Kingdom
Ab78322 is a primary antibody developed for research use. The core function of this product is to detect the target antigen through immunodetection techniques.
Automatically generated - may contain errors
Lab products found in correlation
A0262, by ABclonal (2 mentions)
F3165, by Merck Group (2 mentions)
Ab70892, by Abcam (2 mentions)
Ab32536, by Abcam (2 mentions)
Sc 100736, by Santa Cruz Biotechnology (2 mentions)
A302 089, by Fortis Life Sciences (2 mentions)
A301 813, by Fortis Life Sciences (2 mentions)
A301 034, by Fortis Life Sciences (2 mentions)
Ab6046, by Abcam (2 mentions)
Duo92101, by Merck Group (2 mentions)
Ab92554, by Abcam (2 mentions)
Ba1003, by Boster Bio (1 mentions)
Protein a g agarose, by Merck Group (1 mentions)
Anti ubiquitin, by Merck Group (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti v5, by Cell Signaling Technology (1 mentions)
Ab15580, by Abcam (1 mentions)
Dab staining kit, by Vector Laboratories (1 mentions)
Imager 600, by Cytiva (1 mentions)
Rabbit anti vimentin, by Cell Signaling Technology (1 mentions)
11 protocols using ab78322
1
Immunohistochemical Analysis of KDM5A, p16, and Fbxo22 in Breast Cancer
2
Characterizing KDM5A Ubiquitination in TNBC
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The indicated plasmids (Flag-Fbxo22, HA-KDM5A, and V5-Ubiquitin) were transfected into HEK293T cells. Prior to collection, cells were treated as previously described (Zhang et al. 2019 (link)). The obtained lysates were assayed and immunoprecipitated with protein A/G agarose (Sigma) pre-conjugated with the indicated antibody anti-HA (1:50, #3724, Cell Signaling). In addition, to prevent detection of ubiquitination of the E3 ligase itself and proteins associated with KDM5A, ubiquitination assays were also performed under denaturing conditions. Transfected cells were lysed in lysis buffer and incubated with SDS-PAGE sample buffer at 100 °C for 8 min, followed by incubation with HA antibody for IP and Immunoblotting. Antibodies used for immunoblotting were anti-flag (1:50, F3165, Sigma), anti-HA (1:50, #3724, Cell Signaling), and anti-V5 (1:50, 13,202, Cell Signaling).
Additionally, the ubiquitination level of KDM5A in TNBC cell lines was also examined. The obtained lysates were immunoprecipitated with protein A/G agarose (Sigma) pre-conjugated with the antibody of anti-KDM5A (1:100, ab70892, Abcam) or anti-IgG (1:50, #3900, Cell Signaling). The expression of the relevant proteins was then detected by immunoblotting using the antibodies of mouse anti-KDM5A (1:1000, ab78322, Abcam) and anti-ubiquitin (1:1000, 04–263, Millipore).
Additionally, the ubiquitination level of KDM5A in TNBC cell lines was also examined. The obtained lysates were immunoprecipitated with protein A/G agarose (Sigma) pre-conjugated with the antibody of anti-KDM5A (1:100, ab70892, Abcam) or anti-IgG (1:50, #3900, Cell Signaling). The expression of the relevant proteins was then detected by immunoblotting using the antibodies of mouse anti-KDM5A (1:1000, ab78322, Abcam) and anti-ubiquitin (1:1000, 04–263, Millipore).
3
Immunohistochemical Analysis of Cell Markers
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Paraffin embedded slides were deparaffinized, rehydrated and subjected to antigen-retrieval using citric acid buffer. The endogenous peroxidase was deactivated by H2O2. The slides were blocked using a 10% goat serum solution and incubated with the corresponding primary antibodies overnight at 4°C. The used antibodies were: anti-RBP2 antibody (1:150, Abcam, ab78322), anti-p65 antibody (1:150, Abcam, ab32536) and anti-Ki67 antibody (1:100, Abcam, ab15580). Next, the slides were incubated with a secondary antibody, followed by a colorimetric detection using a DAB staining kit (Vector Laboratories, USA).
4
Protein Expression Analysis in Breast Cancer
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The protein extracts from breast cancer tissues and cells were electro-separated and transferred to PVDF membrane which was then incubated with primary antibodies of mouse anti-KDM5A (1:1000, ab78322, Abcam, UK), rabbit anti-p16 (1:1000, A0262, ABclonal), mouse anti-Fbxo22 (1:1000, sc-100736, Santa Cruz), rabbit anti-E-cadherin (1:1000, #3195, Cell Signaling), rabbit anti-N cadherin (1:1000, #13,116, Cell Signaling), rabbit anti-Vimentin (1:1000, #5741, Cell Signaling), rabbit anti-H3K4me3 (A2357, 1:1000, ABclonal), and rabbit anti-GAPDH (1:1000, #2118, Cell Signaling, internal reference) overnight at 4 °C. HRP-labeled goat anti-mouse (1:10,000, BA1050, Boster, Wuhan, China) or goat anti-rabbit IgG (1:10,000, BA1054, Boster) secondary antibodies were incubated with the membrane for 1 h at ambient temperature. The films were exposed in an Amersham Imager 600 (UK). Gray-scale analysis was then performed using ImageJ.
5
CRISPR-Mediated KDM5A Knockout in U2OS Cells
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KDM5A gene KOs in U2OS cells were generated as previously described (Gong et al., 2017 (link)). The sequence of the single-guide RNA used to edit the gene for human KDM5A was 5′-CGGATGCGGCCGATAAAGCT-3′. Loss of KDM5A protein expression by gene editing in U2OS clones was validated by WB analysis using a mouse anti-KDM5A antibody (ab78322; Abcam).
6
Immunohistochemical Analysis of Bone Marrow
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The mononuclear cells, that were isolated from patient bone-marrow samples, were used to prepare cytospins with glass slides fixed by a polyformaldehyde fixation solution. The samples were stained with anti-RBP2 antibody (1:150, Abcam, ab78322) and anti-p65 antibody (1:150, Abcam, ab32536) overnight at 4°C, followed by an incubation with a horseradish peroxidase-conjugated secondary antibody for 30 min.
7
Immunoprecipitation of Fbxo22 and KDM5A Proteins
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To immunoprecipitate the exogenous proteins, the indicated plasmids (Flag-Fbxo22 and HA-KDM5A) were transfected into HEK293T cells. Cell lysis was carried out in IP lysis buffer (P0013, Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. Cell lysis buffer was obtained by centrifugation at 12,000 g for 20 min at 4 °C. The cell lysis buffer containing 200 μg protein was then incubated with anti-flag antibody (1:50, F3165, Sigma-Aldrich) or anti-HA antibody (1:50, #3724, Cell Signaling, Hercules, CA) for 4 h at 4 °C.
To immunoprecipitate endogenous proteins, the same method as above-described was performed. Antibodies of lysates, KDM5A (1:100, ab70892, Abcam, UK), and IgG (1:50, #3900, Cell Signaling), at a concentration of 1 μg/mg, were added to the cell lysate and incubated overnight at 4 °C. The antibody-protein complexes were then captured with protein A/G Sepharose microbeads (Santa Cruz, CA). The complexes were then subjected to immunoblotting with mouse anti-Fbxo22 (1:1000, sc-100736, Santa Cruz) and mouse anti-KDM5A (1:1000, ab78322, Abcam).
To immunoprecipitate endogenous proteins, the same method as above-described was performed. Antibodies of lysates, KDM5A (1:100, ab70892, Abcam, UK), and IgG (1:50, #3900, Cell Signaling), at a concentration of 1 μg/mg, were added to the cell lysate and incubated overnight at 4 °C. The antibody-protein complexes were then captured with protein A/G Sepharose microbeads (Santa Cruz, CA). The complexes were then subjected to immunoblotting with mouse anti-Fbxo22 (1:1000, sc-100736, Santa Cruz) and mouse anti-KDM5A (1:1000, ab78322, Abcam).
8
Western Blot Analysis of Chromatin Regulators
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Whole-cell extracts were obtained from cells washed with PBS, collected with Laemmli buffer (4% [vol/vol] SDS, 20% [vol/vol] glycerol, and 120 mM Tris-HCl, pH 6.8), and then sonicated with a Diagenode Bioruptor for 10 min followed by 5 min of boiling at 95°C before loading. Samples were resolved by SDS-PAGE and analyzed by standard WB protocols. Signals of the Western blots were detected by standard chemiluminescence (GE Healthcare) and analyzed using a ChemiDoc XRS+ system (Bio-Rad Laboratories). Primary antibodies used were rabbit anti-ZMYND8 (A302-089; Bethyl Laboratories, Inc.), rabbit anti-CHD4 (39289; Active Motif), rabbit anti-HDAC1 (ab19845; Abcam), rabbit anti-HDAC2 (ab7029; Abcam), rabbit anti-MTA2 (A300-395; Bethyl Laboratories, Inc.), mouse anti-KDM5A (ab78322; Abcam), rabbit anti-KDM5B (A301-813; Bethyl Laboratories, Inc.), rabbit anti-KDM5C (A301-034; Bethyl Laboratories, Inc.), rabbit anti-H3 (ab1791; Abcam), rabbit anti-H3K4me3 (9751; Cell Signaling Technology), rabbit anti–β-tubulin (ab6046; Abcam), rabbit anti-GFP (A11122 ; Invitrogen), mouse anti-Flag (F1804; Sigma-Aldrich), rabbit anti-ZNF592 (A301-530; Bethyl Laboratories, Inc.), rabbit anti-ZNF687 (A303-278; Bethyl Laboratories, Inc.), rabbit anti-RBMX (14794; Cell Signaling Technology), rabbit anti-DDB1 (A300-462; Bethyl Laboratories, Inc.), and mouse anti-RAD51 (ab88572; Abcam).
9
Duolink in situ Protein-Protein Interaction Assay
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The Duolink in situ Red starter mouse/rabbit PLA fluorescence assay (Sigma, DUO92101) was performed according to the manufacturer’s instructions. Briefly, proliferating or PMA-induced differentiating K562 cells were attached to slides using cytospin. Cells were washed with 1 × PBS and fixed for 15 min at 4 °C in 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4 °C. The coverslips were blocked with Duolink blocking solution and incubated with CUL4A (Abcam, ab92554, 1:200) and JARID1A (Abcam, ab78322, 1:200) antibodies in Duolink antibody diluent overnight, followed by incubation with PLUS and MINUS PLA probes, ligation, and amplification. The coverslips were then washed and mounted using mounting medium containing DAPI. Images were captured using a wide-field microscope, processed, and analyzed using Zeiss Zen 3.7 and ImageJ software.
10
Proximity Ligation Assay for Protein-Protein Interactions
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The Duolink in situ Red starter mouse/rabbit PLA fluorescence assay (Sigma, DUO92101) was performed according to the manufacturer’s instructions. Briefly, proliferating or PMA-induced differentiating K562 cells were attached to slides using cytospin. Cells were washed with 1×PBS and fixed for 15 min at 4°C in 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4°C. The coverslips were blocked with Duolink blocking solution and incubated with CUL4A (Abcam, ab92554, 1:200) and JARID1A (Abcam, ab78322, 1:200) antibodies in Duolink antibody diluent overnight, followed by incubation with PLUS and MINUS PLA probes, ligation, and amplification. The coverslips were then washed and mounted using mounting medium containing DAPI. Images were captured using a wide-field microscope, processed, and analyzed using Zeiss Zen 3.7 and ImageJ software.
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