Tissue microarrays (TMAs) were purchased from Pantomics (BRC961, BRC962 BRC1501, BRC1502, BRC1503), Richmond, CA, USA). Additional tissue samples of normal breast tissue were produced and processed as previously published57 (link). FFPE cell-blocks were prepared as in Cabezon et al.58 (link). Five micrometer sections were stained with primary antibody against MZF1 (Abcam ab64866, 1:1000, Cambridge, UK) or Lin28A (Abcam ab46020, 1:1000). The sections were scanned and assessed in an ACIS® III Instrument (DAKO). IHC quantification was done by ACIS III-assisted analysis. The stained sections were scanned and brown intensity was quantified with manually placed 40× regions (3 per section/core) and a color threshold customized for MZF1.
Ab64866
Manufactured by Abcam
Sourced in United Kingdom
Ab64866 is a mouse monoclonal antibody that recognizes human Synaptotagmin-1. Synaptotagmin-1 is a calcium-binding protein involved in the regulation of synaptic vesicle exocytosis.
Automatically generated - may contain errors
Lab products found in correlation
Ab32072, by Abcam (2 mentions)
Ab104836, by Abcam (2 mentions)
Ab46020, by Abcam (1 mentions)
Acis 3 instrument, by Agilent Technologies (1 mentions)
Ab59348, by Abcam (1 mentions)
Ab32391, by Abcam (1 mentions)
Ab137675, by Abcam (1 mentions)
Ab126605, by Abcam (1 mentions)
Ab199677, by Abcam (1 mentions)
Ab18197, by Abcam (1 mentions)
Ab32147, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Cell lysis buffer, by Promega (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab9106, by Abcam (1 mentions)
Ab1162, by Abcam (1 mentions)
Ab113687, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
9 protocols using ab64866
1
Quantitative Immunohistochemistry of MZF1 and Lin28A
2
Quantifying Protein Expression in Cells
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Total protein in tissues or cells was extracted with radio immunoprecipitation assay (RIPA) lysis buffer containing phenylmethanesulfonyl fluoride (P0013C; Beyotime Institute of Biotechnology), with the concentration detected by BCA kit. After sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), the protein was electrotransferred onto a polyvinylidene fluoride membrane and blocked with 5% skim milk powder at room temperature for 1 hour. Then, the membrane was probed with diluted primary antibodies to GSK3β (1:5000, ab32391; Abcam), FTO (1:1500, ab126605; Abcam), MZF1 (1:500, ab64866; Abcam,), c‐Myc (1:1000, ab32072; Abcam), CDK2 (1:1000, ab32147; Abcam), CDK4 (1:500, ab137675; Abcam), Ki‐67 (1:1000, ab16667; Abcam), PCNA (1:1000, ab18197; Abcam), Bax (1:1000, ab199677; Abcam), Bcl‐2 (1:500, ab59348; Abcam) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (ab9485, 1:2500; Abcam) at 4°C overnight. Next day, the membrane was re‐probed with horseradish peroxidase (HRP)‐labelled secondary antibody of goat anti‐rabbit antibody to immunoglobulin G (IgG) for 1 hour and visualized by enhanced chemiluminescence (ECL) kit (BB‐3501, Ameshame; Chiltem). Finally, Quantity One v4.6.2 software was used to quantify the grey levels of each band in the Western blot image. GAPDH served as an internal reference.
3
Generating Antibodies for MZF1 Protein
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The peptide corresponding to MZF-uPEP (METRWGTDGVLMTAVIGAGSC) was synthesized, and coupled to keyhole limpet hemocyanin using chemical crosslinker glutaraldehyde. Rabbit anti-MZF1-uPEP polyclonal antibody was prepared by immunizing New Zealand rabbit with synthesized peptide, purified by persulfate, Sephadex G25 and DEAE-Sephadex G100 (ABclonal Biotechnology Co., Ltd, Wuhan, China), and validated by antigen peptide or fusion protein recognition. Tissue or cellular protein was extracted with 1× cell lysis buffer (Promega, Madison, WI). Western blot was performed as previously described 16 (link)-20 (link), with antibodies for MZF1 (ab64866), HK2 (ab104836), PGK1 (ab113687), phosphorylated AKT (p-AKT, ab38449), AKT (ab8805, Abcam Inc., Cambridge, MA), YY1 (D3D4Q, Cell Signaling Technology, Inc., Danvers, MA), upstream transcription factor 2 (USF2, ab125184), GFP (ab290), Flag (ab1162), Myc (ab9106), or β-actin (ab6276, Abcam Inc.).
4
Western Blot Analysis of Cellular Signaling
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Immunoblotting was carried out as previously described [13 (link)]. Antibodies against PLK3 (4896), PCNA (2586), p-STAT3 (Tyr705, 9145), p-STAT3 (Ser727, 9134), p-p65 (3033) and total STAT3 (9139) were purchased from Cell Signaling Technology. Antibodies against HK2 (ab104836), HSP90 (ab13492), HIF-1α (ab2185), c-Myc (ab32072), MZF1 (ab64866) and GAPDH (ab9484) were purchased from Abcam.
5
Protein Extraction and Western Blotting
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SH-SY5Y cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) to extract proteins. After the protein concentration was measured using BCA kits (Beyotime), the corresponding volume of protein was added into and mixed with the loading buffer (Beyotime). The protein was denatured by a boiling water bath for 3 min. Electrophoresis was initially run at 80V for 30 min, and then switched to 120V for 1-2 hr after bromophenol blue entered the separation gel. The protein was blotted onto membranes in an ice bath for 60 min with a current of 300 mA. After membrane transfer, the membrane was rinsed in washing solution for 1-2 min, and then it was placed in the sealing solution at room temperature for 60 min, or for sealing overnight at 4°C. At room temperature, primary antibody against MZF1 (ab64866, 1:1000), RBM3 (ab134946, 1:1000), or β-actin (ab8226, 1 µg/mL, Abcam, Cambridge, MA, USA) was incubated with the membrane on a rotary shaker for 1 hr, and washing solution was used to wash the membrane, 10 min each time. The membrane was then incubated with secondary antibody goat anti-rabbit IgG for 1 hr at room temperature, and washed for 3 times, 10 min each time. Chemiluminescence imaging system (Gel Doc XR, Bio-Rad) was used for detection after the developer solution was dropped onto the membrane.
6
Immunohistochemical Profiling of Tumor Markers
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Immunohistochemical staining was performed as previously described 16 (link)-18 (link), with antibodies specific for Ki-67 (ab92742; 1:100 dilution), MZF1-uPEP (1:200 dilution), YY1 (ab38422; 1:200 dilution), or MZF1 (ab64866, Abcam Inc.; 1:200 dilution). The immunostaining specificity was validated by neutralizing antigen peptide incubation and IgG isotype control. The reactivity degree was assessed by at least two pathologists without knowledge of tumor groups.
7
Immunofluorescence Assay for MZF1
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IF was performed according a standard protocol. Briefly, BGC823 and GES1 cells were seeded onto slides. The slides were fixed with 4% paraformaldehyde for 15 min. Then, the slides were blocked with 5% non-fat milk for 1 h at room temperature and incubated with the primary antibody against MZF1 (ab64866, Abcam, Cambridge, UK) overnight at 4°C. The next day, the slides were incubated with cyanine-conjugated secondary antibodies for 1 h in the dark. 4',6-diamidino-2-phenylindole (DAPI) staining (1:5000 vol/vol of a 5 mg/ml stock) was performed to stain the DNA. Confocal images were obtained using a confocal microscope (Olympus FV1000: Olympus Corporation, Tokyo, Japan).
8
Immunohistochemical Quantification of MZF1 Expression
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Rabbit polyclonal antibody against MZF1 at a dilution of 1:200 (ab64866, Abcam, Cambridge, UK) was used according to a previously reported in IHC staining protocol [4] . Immunostaining was developed using the universal immuno-peroxidase polymer anti-mouse/rabbit IHC staining reagent (ZSGB-BIO, China), and haematoxylin was used for counter staining. Antibody was omitted in the negative control slides in this study. IHC staining for MZF1 was scored based on a semi-quantitative scale (-to +++) system. Cells positively stained for MZF1 showed variable degrees of nuclear and cytoplasmic staining in the tissues. The scoring was based on the staining intensity (0 = none; 1 = mild; 2 = moderate; and 3 = intense) and percentage of positive cells (0 = 0%; 1 = 1%-25%; 2 = 26%-50%; 3 = 51%-75%; 4 = 76%-100%). The final score of each sample (0-12) was obtained by multiplying the staining intensity with the percentage of positive cells, and tissues were ultimately defined as negative (-), score 0; weak expression (+), score 1-4; moderate expression (++), score 5-8; and strong expression (+++), scored ≥ 9. Tissue samples scoring (+) to (+++) were considered "positive", and samples scoring (-) were defined as "negative".
9
Quantifying Protein Expression Using Western Blot
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Protein lysates were obtained using RIPA lysis buffer supplemented with protease inhibitor (Roche, USA) according to standard protocols. BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL) was used to measure protein concentration in the samples according to the manufacturer's instructions. The protein samples were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked with PBST (1× PBS containing 0.1% Tween-20) with 5% non-fat milk. Then, the membranes were incubated with rabbit polyclonal antibody against MZF1 (1:1000, ab64866, Abcam, Cambridge, UK) or anti-GAPDH antibody (1:5000, Kangchen lnc. Shanghai, China) overnight. Finally, the membranes were incubated with horseradish peroxidase (HRP)conjugated secondary antibodies incubated for 1 h at room temperature. Images of the membraned were obtained using the Odyssey Sa Infrared Image System (Li-COR Corp. Lincoln, Nebraska USA).
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