Antibodies were purchased from the following companies: Mst1 (#3682; WB, 1:1000; IHC, 1:200), phospho-Mst1-Thr183 (#3681; WB, 1:1000; IHC, 1:200), phospho-Nur77-Ser351 (#5095; WB, 1:1000), Flag (#14793; WB, 1:1000), Myc (#2276; WB, 1:1000; IF, 1:100) (all from Cell Signalling Technology), phosphothreonine (#P6623; WB, 1:500), phosphoserine (#P5747; WB, 1:1000) (all from Millipore Sigma), Nur77 (#ab48789, Abcam; WB, 1:1000; IHC, 1:200), HOXA10 (#sc-17158; WB, 1:1000), and phospho-Nur77-Ser341 (#sc-16991; WB, 1:1000) (all from Santa Cruz Biotechnology, Inc.), β3-integrin (#BS3660; WB, 1:1000), GAPDH (#AP0063; WB, 1:2000) (all from Bioworld Technology, Inc.), FAK (#CY5464; WB, 1:1000), phospho-FKA-Y397 (#CY5464; WB, 1:1000) (all from Abways Technology, Inc.). The phosphorylation state-specific antibody against phospho-Nur77-Thr366 was provided by Nanjing Peptide Biotech (WB, 1:400). As brief, the synthetic phosphorylated peptide ANLLTpSLVRA was subjected to immunization of rabbits, following with a two-step affinity purification with a non-phosphorylated peptide column and a phosphorylated peptide column to get the polyclonal antibody against phospho-Nur77-Thr366.
Hoxa10
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HOXA10 is a protein coding gene that plays a role in the regulation of gene expression, embryonic development, and cell differentiation. It is an important transcription factor involved in the development of the urogenital system and the regulation of endometrial receptivity during the menstrual cycle. The product is suitable for various research applications, including studies on embryonic development, reproductive biology, and gene regulation.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Bioworld Technology (3 mentions)
Enhanced chemiluminescence kit, by Merck Group (2 mentions)
Nur77, by Abcam (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Phosphoserine, by Merck Group (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ubiquitin, by Abcam (1 mentions)
Senp2, by Abcepta (1 mentions)
Goat anti rabbit secondary antibody, by Bioworld Technology (1 mentions)
Sumo1, by Abcam (1 mentions)
Senp1, by Abcam (1 mentions)
Lamin b1, by Bioworld Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
P y705 stat3, by Bioworld Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Mettl3, by Abcam (1 mentions)
Dab substrate, by Beyotime (1 mentions)
Integrin β3, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Nf κb p50, by Santa Cruz Biotechnology (1 mentions)
7 protocols using hoxa10
1
Phosphorylation-specific Antibody Generation
2
Phosphoproteomic Analysis of Breast Cancer Cells
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Breast cancer cell lines were lysed and loaded onto NuPAGE® gels (Invitrogen). After electrophoresis was completed, proteins were transferred onto a pre-wet nitrocellulose membrane (Life Technologies). Membranes were rinsed with water twice, and then were blocked 1 hour at room temperature with gentle agitation in freshly prepared Tris buffered saline (TBS) and 1% Tween 20 (TBST) containing either 2% milk or 2% BSA. To enrich phosphopeptides in breast cancer cell lines we utilized phosphopeptides enrichment columns (Clontech). Membranes were incubated with antibodies against HER2, AKT, pAKT, pHER3, pHER2/ErbB2 (Tyr1221/1222), NFKB (Cell Signaling) and HOXA10 (Santa Cruz) diluted in TBST overnight at 40C with agitation. After washing the membranes three times with TBST, they were incubated with the appropriate secondary antibody (Anti Rabbit or Anti Goat IgG) and incubated for 1 hour at room temperature and detected using chemiluminescence reagents (Amersham, ECL Western Blotting detection reagents). Western blot images were quantified using Alpha Ease FCTM (Alpha Innotech Corporation) imager software (Figure S1 ).
3
SUMO Regulation of HOXA10 Expression
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Tissues and cells were homogenized in whole-cell lysis buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 1.0% NP-40) containing a protease inhibitor cocktail and 20 mM N-ethylmaleimide to prevent SUMO de-conjugation. Immunoblotting was performed with primary antibodies against HOXA10 (1 : 1000; Santa Cruz, Santa Cruz, CA, USA), SUMO1 (1 : 1000; Abcam, Cambridge, CA, USA), SENP1 (1 : 1000; Abcam), SENP2 (1 : 1000; Abgent, San Diego, CA, USA), Flag (1 : 1000; Sigma), Myc (1 : 5000; Invitrogen, Carlsbad, CA, USA), Lamin B1 (1 : 1000; Bioworld, St Louis Park, MN, USA), HSP90B (1 : 1000; Bioworld), ubiquitin (1 : 1000, Abcam), acetylated lysine (1 : 1000; CST, Danvers, MA, USA) and GAPDH (1 : 10 000; Bioworld), followed by donkey anti-goat or goat anti-rabbit secondary antibody conjugated with HRP. Detection was performed using an enhanced chemiluminescence kit (Millipore, Billerica, USA).
4
Protein Expression Analysis Protocol
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Tissues and cells were homogenized in whole-cell lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl and 1.0% NP-40) containing phosphatase and protease inhibitors. Immunoblotting was performed with primary antibodies against HOXA10 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-10 (1:1000; Bioworld, St. Louis Park, MN, USA), STAT3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), p(Y705)-STAT3 (1:1000; Bioworld), or GAPDH (1:10,000; Bioworld), followed by a donkey anti-goat or goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP). Detection was performed using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA).
5
Immunohistochemical Analysis of Protein Expression
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The immunohistochemistry (IHC) analysis was performed using a Dako K5007 kit (Dako, Denmark) according to the manual instructions. The staining intensity was scored as (0 = negative, 1 = weak, 2 = moderate, 3 = strong) according to the expression level and the staining area was scored as (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–100%) based on the percentage of positive cells. The immunostaining score (IS) was calculated by adding the score of staining intensity and area according to the method of Yuan et al. [34 ]. The positive specimen for HOXA10 and E-cadherin individually was defined as an IS≥3, while negative staining with an IS < 3. The primary antibodies used in immunohistochemistry included: HOXA10 (1:100, Santa Cruz), E-cadherin (1:400, CST), N-cadherin (1:125, CST), Vimentin (1:200, CST), METTL3 (1:500, Abcam).
6
Immunohistochemical Analysis of MLL1 and HOXA10 in Human Endometrium
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Immunohistochemistry staining was performed on paraffin sections with antibody against MLL1 (1:200; Abcam) and antibody against HOXA10 (1:200; Santa cruz). Immunostaining was performed as previous research described. Endometrial tissues were fixed by 4% paraformaldehyde and embedded in paraffin. Then the sections were deparaffinized and rehydrated in graded ethanol, and antigen retrieval was performed. Sections were then treated with 3% hydrogen peroxide for 5 minutes to inhibit endogenous peroxidase activity. After blocking for 30 minutes, sections were incubated overnight at 4°C with primary antibodies. On the next day, sections were incubated with peroxidase‐labelled anti‐rabbit IgG for 30 minutes. Finally, all slides were incubated with DAB‐Substrate (Beyotime) and counterstained in haematoxylin before they were dehydrated and mounted. IgG antibody was used in human endometrium as a negative control (Data were not shown). Ten fields were selected for each immunohistochemical section, and all these slides were used to conduct semi‐quantitative histologic scoring (H‐score) analysis by ImageJ.
7
Antibody Procurement for Cell Analysis
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Antibodies for TPPP (sc-98687), α-tubulin (sc-8035), β-tubulin (sc-5274), WNT4 (sc-376279), LIF (sc-20087) , IHH (sc-13088), Fz-2 (sc-68328), HoxA10 (sc-17159), Integrin-β3 (sc-52685), NF-κB p50 (sc-53744), Cytokeratin (sc-57004), Vimentin (sc-32322), ERα (sc-543), PR (sc-538), JNK (sc-572) and GAPDH (sc-32233) were procured from Santa Cruz Biotechnology. Antibody for NF-κB p65 (#8242) was procured from Cell Signaling Technology and STAT3 (610189) was procured from BD Biosciences.
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