Protein was extracted using Mammalian Protein Extraction Reagent (Thermo Fisher, Waltham, Mass) with protease and phosphatase inhibitors and electrophoresed on NuPage Novex 4–12% Bis-Tris Protein Gels, transferred to PVDF membranes, and blocked for one hour with 5% w/v BSA or 5% nonfat milk in 1X TBS and 0.1% Tween-20. Immunoblots were performed using rabbit anti-IQGAP1 (Abcam ab86064, Abcam, Cambridge, Mass), anti-pERK1/2 (Cell Signaling CST9101S, Cell Signaling, Danvers, Mass), and anti-ERK1/2 (Cell Signaling CST9102) polyclonal, and mouse anti-GAPDH monoclonal (Abcam ab8245) antibodies. Proteins were detected using the Amersham ECL reagent (GE Healthcare Life Sciences, Pittsburgh, Pa) on HyBlot CL film (Denville Scientific, Holliston, Mass). Densitometric analysis of protein expression was performed using ImageLab software (BioRad, Hercules, Calif).
Ab86064
Manufactured by Abcam
Sourced in United States
Ab86064 is a laboratory equipment product. It is a core functional component used in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Amersham ecl reagent, by GE Healthcare (1 mentions)
Hyblot cl film, by Thomas Scientific (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab9332, by Abcam (1 mentions)
Alkbh5, by Proteintech (1 mentions)
Nanog, by Proteintech (1 mentions)
Mettl14, by Proteintech (1 mentions)
Mettl3, by Proteintech (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using ab86064
1
Western Blot Analysis of IQGAP1, pERK1/2, and ERK1/2
2
Western Blot Antibody Panel for Stem Cell Analysis
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The procedures of western blot assay have been described previously.27 Antibodies that recognize AMD1 (11052‐1‐AP), Tubulin (11224‐1‐AP), NANOG (14295‐1‐AP), SOX2 (11064‐1‐AP), KLF4 (11880‐1‐AP), OCT4 (11263‐1‐AP), METTL3 (15073‐1‐AP), METTL14 (26158‐1‐AP), FTO (27226‐1‐AP), and ALKBH5 (16837‐1‐AP) were purchased from Proteintech (Wuhan, China). Antibodies that recognize IQGAP1 (ab86064), phosphoserine (ab9332), phosphothreonine (ab9338), and SPM (ab7318) were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies were obtained from Beyotime Biotechnology (Shanghai, China).
3
Immunohistochemical Analysis of IQGAP1 in Gliomas
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3 paired paraffin-embedded GBMLGG and counterpart normal adjacent tissues were collected from the second hospital of Dalian Medical University. Using IHC, the protein expression levels of IQGAP1 were found in 4-μm tissue slices. Sections were rehydrated in varying alcohols after being deparaffinized in xylene. Sections underwent a 20-minute, 95°C pretreatment in citrate buffer (0.01 mol/L citric acid, pH 6.0). They were then submerged in PBS containing 3% H2O2 for ten minutes at room temperature. Following treatment with 10% normal goat serum in PBS for 30 minutes at room temperature, the tissue slices were incubated with rabbit polyclonal anti-IQGAP1 antibodies (1:1000 dilution) (ab86064, Abcam) for an entire night at 4°C. Following a PBS washing, the sections were treated with 3,30-diaminobenzidine chromogen for five minutes at room temperature and incubated for twenty minutes with biotinylated goat anti-rabbit IgG. Sections were then counterstained for six minutes using hematoxylin. The second hospital of Dalian Medical University’s Ethics Committee accepted the portion of the study that involved experimentation on human tissues. We acquired informed consent from each and every participant.
4
IP-MS Approach for Interacting Proteins
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For IP, cell lysis was performed using Pierce IP Lysis Buffer (Thermo Fisher Scientific) in accordance with the instruction. Antibody against FTO (1:50, #31687, Cell Signaling Technology) or against IQGAP1 (1:100, ab86064, Abcam) was added to lysates with protein A/G beads and incubated overnight at 4℃. After centrifuged, the deposit was collected and washed with lysis buffer for three times. After resuspended in RIPA buffer, it was boiled for 5 min. The boiled compounds were centrifuged again, and the supernatant was used for following western blot and mass spectrometry (MS) assay. MS assay was to detect the potential interacting proteins as described previously.29 Open search strategy for higher confidence was applied to identify potential interacting proteins and various modifications as described.30
5
Immunofluorescence Visualization of IQGAP1
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GC cells were fixed with 4% paraformaldehyde and treated with Triton X-100. Cells were blocked with donkey serum at 37 °C for 30 min and then incubated with IQGAP1 antibodies (1:100; ab86064, Abcam) overnight at 4 °C. The next day, cells were incubated with a fluorescent secondary antibody (Thermo Fisher) and then stained with DAPI. Finally, protein fluorescence pictures were captured and evaluated using a fluorescence microscope.
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