ZFL cells were transfected in six-well plates with miR-145 mimic (final concentration 100 nM), miR-145 hairpin inhibitor (final concentration 100 nM) or miR-control (final concentration 100 nM). After transfection, cells were cultured for 48 hours. Intermediate samples were collected and analyzed by western blot to assess GrnA and MET expression. The lysates were hybridized with the following primary antibodies: the polyclonal anti-GrnA antibody (1:1000), which was produced using the 4MAPS peptide EWEDHKQKKPETQRTTTRPTG (corresponding to residues 244–264 of GrnA) to immunize BALB/c mice (LTK Biolab Incorporation, Taiwan); Met antibody (1:1000, sc-10; Santa Cruz Biotechnologies, USA); and GAPDH antibody (1:1000; AnaSpec, USA).
Sc 10
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Sc-10 is a laboratory instrument designed for the preparation and analysis of biological samples. It is a core piece of equipment used in various life science research applications.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh antibody, by AnaSpec (2 mentions)
Sc 138, by Santa Cruz Biotechnology (1 mentions)
Ab32562, by Abcam (1 mentions)
Ab 11779, by Abcam (1 mentions)
Glycerol mounting medium, by Agilent Technologies (1 mentions)
Anti c met, by Santa Cruz Biotechnology (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (1 mentions)
Dako kit, by Agilent Technologies (1 mentions)
5 protocols using sc 10
1
Assess miR-145 Effects on GrnA and MET
2
Production and Purification of Phospho-specific Antibodies
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Mouse monoclonal antibodies against GST and Myc-epitope tag were purchased from Santa Cruz (cat # sc-138 and sc-10, respectively). Polyclonal antibodies against phosphorylated PAPC were raised by immunizing rabbits with a synthetic peptide ‘MGHI(pS)TKD(pS)GKGD’. ‘(pS)’ indicates a phosphorylated serine residue. The antisera were affinity-purified with the phosphorylated peptide, followed by absorption of the non-phosphorylated peptide ‘MGHISTKDSGKGD’.
3
Immunofluorescence Staining of Cell Markers
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Cells grown on glass coverslips were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT), and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Fixed cells were blocked with 5% BSA in PBS for 30 min, incubated overnight at 4°C with primary Ab, washed, incubated 1 h at RT with secondary Ab, and counterstained with DAPI (Pharmingen). Coverslips were mounted with glycerol mounting medium (Dako) and sealed with clear nail polish. Fluorescence images were obtained by confocal immunofluorescence microscopy (model TCS SP8; Leica).
Primary Abs used for immunofluorescence staining were anti-GD2 (mouse, Clone 14.G2a, #554272, BD Biosciences), anti-GD3 (mouse, clone R24, ab 11779, Abcam), anti-EGFR (rabbit, ab32562; Abcam), and anti-c-Met (rabbit, sc-10, Santa Cruz). Secondary Abs used were Alexa555-conjugated donkey anti-mouse IgG and donkey anti-rabbit IgG (Jackson ImmunoResearch) and Alexa488-conjugated donkey anti-rabbit IgG and donkey anti-mouse IgG (Invitrogen).
Primary Abs used for immunofluorescence staining were anti-GD2 (mouse, Clone 14.G2a, #554272, BD Biosciences), anti-GD3 (mouse, clone R24, ab 11779, Abcam), anti-EGFR (rabbit, ab32562; Abcam), and anti-c-Met (rabbit, sc-10, Santa Cruz). Secondary Abs used were Alexa555-conjugated donkey anti-mouse IgG and donkey anti-rabbit IgG (Jackson ImmunoResearch) and Alexa488-conjugated donkey anti-rabbit IgG and donkey anti-mouse IgG (Invitrogen).
4
Immunohistochemical Analysis of CEACAM5, EpCAM, and c-Met
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Based on hematoxylin–eosin-stained slides, a representative FFPE tissue block containing tumor and normal tissue from each patient was chosen by a board-certified pathologist (A.F.S.). After sectioning the FFPE blocks in 4 µm slides, these were mounted on adhesive slides (Starfrost), deparaffinized using xylene and rehydrated in decreasing concentrations of ethanol. Subsequently, slides were rinsed with distilled (DI) water and endogenous peroxidase was blocked with 0.3% hydrogen peroxidase (Merck Millipore) for 20 minutes. Slides were rinsed with DI water and antigen retrieval was performed in the DAKO PT LINK, Target Retrieval Solution pH 6.0 at 95°C for 10 minutes. After rinsing with phosphate buffered saline, slides were stained with predetermined dilutions using monoclonal antibodies (mAb) against CEACAM5 (clone CI-P83-1, SC-23928 from Santa Cruz Biotechnology, 0.2 µg/mL, dilution 1:2,500), EpCAM (clone MOC-31, Acris Antibodies, dilution 1:10,000) and a polyclonal antibody against c-Met (polyclonal rabbit, Santa Cruz SC-10, 1 µg/mL, dilution 1:100). After overnight incubation with the primary antibodies, slides were incubated with the secondary antibody (EnVision antimouse horseradish peroxidase [DAKO]) for 30 minutes, followed by diaminobenzidine solution (DAB+; DAKO Kit). All slides were counterstained with hematoxylin, dehydrated and finally mounted with pertex.
5
Immunohistochemistry and Western Blot Analysis of Liver Tissue
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For immunohistochemistry, 10% neutral buffered formalin-fixed liver tissues were sectioned and hybridized with a PCNA antibody (PC10; Abcam, Cambridge, UK). The liver samples were collected and analyzed by Western blot to assess GrnA, HGF, and c-met expression. The lysates were hybridized with the following primary antibodies: polyclonal anti-GrnA antibody (1:1000), which was produced by immunizing BALB/c mice with 4MAPS peptide EWEDHKQKKPETQRTTTRPTG (corresponding to residues 244–264 of GrnA) (LTK Biolab Incorporation, New Taipei City, Taiwan), HGF antibody (1:1000; Aviva Systems Biology, San Diego, CA, USA), c-met antibody (1:1000, sc-10; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and GAPDH antibody (1:1000; AnaSpec, Fremont, CA, USA).
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