After extracting total proteins with RIPA Lysis Buffer (Beyotime), the same amount of protein was separated by 10% SDS-PAGE gel and then transferred onto PVDF membranes (Invitrogen). The membrane was incubated with anti-IGF1R (1:1,000, ab263907, Abcam), anti-p-AKT (1:25,000, ab81283, Abcam), anti-AKT (1:500, ab8805, Abcam), anti-cyclin D1 (1:200, ab16663, Abcam), anti-GLUT1 (1:2,500, ab14683, Abcam), anti-YTHDC2 (1:1,000, ab220160, Abcam), or anti-β-actin (1:5,000, ab8226, Abcam) followed by hatching with Goat anti-Rabbit (1:50,000, ab205718, Abcam) or Goat-anti-Mouse (1:5,000, ab205719, Abcam). Protein signals were visualized with the BeyoECL Plus Kit (Beyotime).
Ab14683
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab14683 is a monoclonal antibody product from Abcam. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab205718, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab23750, by Abcam (2 mentions)
Sc 365684, by Santa Cruz Biotechnology (2 mentions)
Ab5694, by Abcam (2 mentions)
Ab47010, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab205719, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab220160, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ab81283, by Abcam (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Ab19304, by Abcam (1 mentions)
Ab175933, by Abcam (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Polyperoxidase anti rabbit igg, by ZSGB-BIO (1 mentions)
13 protocols using ab14683
1
Western blot analysis of IGF1R pathway
2
Immunohistochemical and Immunofluorescence Analysis of Tumor Markers
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Tumor tissues were fixed with 4% paraformaldehyde and then sectioned into 4 μm of sections. IHC was performed according to protocols of the manufacturor. The sections were incubated with rabbit anti-MMP2, MMP9, p-ATM, GLUT1, PKM2 and TGFβ1 polyclonal antibody (1:200, Bioworld) overnight at 4 °C. Then, the sections were sequentially incubated with polyperoxidase-anti-rabbit IgG (ZSBiO) for 30 min at 37 °C, then stained with diaminobenizidine.
Immunofluorescence staining was done following the standard protocol as described previously [16 ]. The primary antibodies specifically against FN (ab23750, abcam,1:200), α-SMA (ab5694, abcam,1:200), ATM (ab47575, abcam, 1:200), p-ATM (ab19304, abcam, 1:200), γH2AX (5883, CST, 1:200), 53BP1 (ab175933, abcam, 1:200), GLUT1 (ab14683, abcam, 1:200), PKM2 (sc365684, Santa Cruz, 1:150) were used. Normal rabbit IgG was the negative control. IHC and IF images were captured using a Nikon Eclipse 80i microscope (Tokyo, Japan).
Immunofluorescence staining was done following the standard protocol as described previously [16 ]. The primary antibodies specifically against FN (ab23750, abcam,1:200), α-SMA (ab5694, abcam,1:200), ATM (ab47575, abcam, 1:200), p-ATM (ab19304, abcam, 1:200), γH2AX (5883, CST, 1:200), 53BP1 (ab175933, abcam, 1:200), GLUT1 (ab14683, abcam, 1:200), PKM2 (sc365684, Santa Cruz, 1:150) were used. Normal rabbit IgG was the negative control. IHC and IF images were captured using a Nikon Eclipse 80i microscope (Tokyo, Japan).
3
Western Blotting Analysis of Cellular Proteins
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Western blotting analysis was performed as described previously [11 (link)]. Briefly, total cell proteins were obtained using RIPA lysis buffer (P0013B, Beyotime, China), quantified with the BCA protein assay kit (P0012, Beyotime). 50 μg of total proteins were separately electrophoresed in 8%–12% SDS-PAGE gel, subsequently incubated with appropriate primary antibodies as followings: FN (ab23750, abcam,1:1000), FAP (ab53066, abcam,1:1000), α-SMA (ab5694, abcam,1:1000), ATM (2873, CST, 1:1000), p-ATM (5883, CST, 1:1000), γH2AX (9718, CST, 1:1000), CHK2-T68 (ab32148, abcam, 1:1000), Na+/K+ ATPase (ab58457, abcam, 1:800), Hsp90 (ab13492, abcam, 1:800), AKT (4685, CST, 1:1000), p-AKT (12694 s, CST, 1:1000), GLUT1 (ab14683, abcam, 1:500), p-ST/Q (6966 s, CST, 1:1000), PKM2 (sc365684, Santa Cruz, 1:500), MCT4 (ab74109,1:1000), MCT1 (ab90582,1:1000) TGFβ1 (ab675195, abcam, 1:1000), P38 (bs4635, bioworld, 1:1000), p-P38 (bs3566, bioworld, 1:1000), MMP2 (ab92538, abcam, 1:800), and MMP9 (ab76003, abcam, 1:800), GLUT3 (ab41525,1:800), HK2 (ab104836,1:800), HPI (ab86950,1:1000), LDHA (ab101562,1:1000). The appropriate horseradish peroxidase (HRP)-conjugated anti-mouse or rabbit IgG (ZSGBBIO, China) was used as secondary antibodies. The protein bands were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Tokyo, Japan).
4
Protein Extraction and Western Blot Analysis
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The standard protocol was used to extract proteins and perform Western-blot assays as previously described [37 (link)]. Antibodies against Glut1 (ab14683, Abcam; 1:1,000), anti-LIF (AF-250-NA, R & D; 1:1000), anti-Na/K ATPase (ab58479, Abcam, 1:2,000), anti-p-AKT (4051, Cell Signaling; 1:2,000), anti-AKT (SC-1618, Santa Cruz; 1:2,000), and anti-β-actin (A5441, Sigma; 1:125,000) were used in this study.
5
Quantitative Protein Expression Analysis
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Cells in each group were lysed with RIPA lysate, and protein concentrations were detected by a BCA kit. The protein was separated by SDS‐PAGE and transferred to a PVDF membrane. The membrane was blocked at room temperature for 1 h and incubated overnight at 4°C with anti‐GLUT1 (ab14683, 1:100, Abcam), LDHA (ab47010, 1:100, Abcam), Ki67 (ab15580, 1:100, Abcam), MMP‐2 (ab92536, 1:100, Abcam), MMP‐9 (ab283575, 1:100, Abcam), and GAPDH (ab8245, 1:100, Abcam). Wash the membrane twice the next day, add diluted enzyme‐labeled secondary antibody, and incubate at room temperature for 1 h. The chemiluminescence reagent was added to develop the protein, and the image was collected in the gel imaging system. The protein level was analyzed by Image J software. GAPDH was used as the internal reference to calculate the relative expression of the protein.
6
Immunolabeling of Tissue Sections
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Sections were air-dried overnight at room temperature, followed by rehydration with phosphate-buffered saline (PBS; Sigma). The sections were incubated for 1.5 h in a blocking buffer containing 10% normal goat serum (Vector laboratories, Newark, CA, USA) diluted in PBST (PBS with 0.1% Triton X-100). For immunofluorescent labelling, the slides were incubated overnight at 4 °C in related primary antibodies (rabbit-anti-IBA1 1:200, 019-19741, WAKO; mouse-anti-ED1 1:100, MCA341GA, Bio-Rad; rabbit-anti-HIF1a 1:100, ab82832, Abcam; rabbit-anti-pimonidazole 1:200, HP3-1000, HPI Inc.; rabbit-anti-GLUT1 1:100, ab14683, Abcam; rabbit-anti-iNOS 1:100, PA1-036, Invitrogen; mouse-anti-3NT 1:100, ab61392, Abcam) diluted in a blocking buffer and then for 1 h in corresponding secondary antibodies diluted 1:200 (AlexFluor546-conjugated goat-anti-rabbit IgG, A11035; AlexFluor488-conjugated goat-anti-mouse IgG, A11001; Invitrogen), before being mounted with a DAPI-containing mounting medium (Biotium, Fremont, CA, USA). For labelling by immunohistochemistry, biotin-conjugated secondary antibodies (goat-anti-rabbit, BA1000, Vector laboratories) were used, and the slides were incubated in a biotin-streptavidin amplification solution (ABC kit; Vector laboratories) for 1 h, followed by a diaminobenzidine (DAB; Vector laboratories) reaction.
7
Immunohistochemical Analysis of GLUT-1 and HIF-1α in Laryngeal and Hypopharyngeal Carcinoma
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The expression of GLUT-1 and HIF-1 was detected by immunohistochemistry in 55 cases of laryngeal and hypopharyngeal carcinoma, and 20 cases of vocal cord polyps. Paraffin wax blocks of formalin-fixed biopsies specimens were obtained from the predominant lesions in each subject. Serial sections (4 µm) subjected to immunohistological staining were fixed with 3% H2O2 to block endogenous peroxidase activity, and treated with antigen retrieval solution for 15 min. The sections were incubated with primary monoclonal anti-GLUT-1 (dilution, 1:100; catalog no., ab14683; Abcam, Cambridge, UK) or monoclonal anti-HIF-1α (dilution, 1:200; catalog no., ab51608; Abcam) antibody for 30 min at 36−38 °C, followed by incubation with a secondary antibody (K5007, Dako) for 15 min at 20−25 °C. The final reaction product was developed by exposure to 0.03% diaminobenzidine, and the nuclei were counterstained with hematoxylin.
8
Protein Extraction from Cancer Cells
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The protein extraction protocol from cancer cells was previously reported [30 (link)]. Briefly, cells were collected and lysed using Radio Immunoprecipitation Assay (RIPA) protein extraction reagent (Beyotime) with a protease inhibitor cocktail (Roche, IN, USA). Equal amounts of protein lysate were quantified using BCA kit (Thermo Fisher, USA), separated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then sealed with 5% non-fat milk for 1 hour, and incubated with primary antibodies (Abcam, Cambridge, UK) against: FUS (ab84078, 1/1000), SLC10A1 (ab131084, 1/1000), β-actin (ab8227, 1/1000), GLUT1 (ab14683, 1/2500), HK2 (ab227198, 1/5000), LDHA (ab47010, 1/1000), PGK1 (ab154613, 1/1000) overnight at 4°C, with GAPDH (ab9485, 1/2500) as control. After washing, the membrane was incubated with goat anti-rabbit secondary antibody (ab96899, 1/1000, Abcam, Cambridge, UK) at 37°C for 1 hour. The protein bands were finally detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) according to manufacturer's instructions.
9
Western Blot Analysis of Metabolic Proteins
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Proteins were extracted with RIPA buffer (Beyotime). Then, the proteins were separated via electrophoresis using SDS-PAGE gels and then transferred onto PVDF membranes. After blocking, membranes were incubated with primary antibody and HRP-labeled secondary antibody. Then, protein signals were visualized using an ECL reagent (Solarbio, Beijing, China). All antibodies are listed as below: anti-LDHA (1:2000, ab125683, Abcam, Cambridge, MA, USA), anti-GLUT1 (1:2500, ab14683, Abcam), anti-HK2 (1:1000, ab209847, Abcam), anti-c-Myc (1:1000, ab32072, Abcam), anti-HIPK2 (1:1000, 55408–1-AP, Proteintech, Rosemont, IL, USA), anti-CD61 (1:1000, 25682–1-AP, Proteintech), anti-CD81 (1:6000, 66866–1-Ig, Proteintech), anti-TSG101 (1:5000, 28283–1-AP, Proteintech), anti-GM130 (1:5000, 11308–1-AP, Proteintech), anti-β-actin (1:1000, ab8227, Abcam), Goat anti-rabbit IgG (1:50,000, ab205718, Abcam) and Goat anti-mouse IgG (1:10,000, ab6789, Abcam).
10
Osteoclast Protein Expression Analysis
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Osteoclasts were sonicated in lysis buffer (6.2 M urea, 10% glycerol, 5 mM dithiothreitol, 1% sodium dodecyl sulphate, protease inhibitors) before cell extract was separated by 8% SDS-PAGE and transferred onto a PVDF membrane. Membranes were probed with primary antibodies specific for HIF-1α (clone 54, 1:1000; BD Biosciences, Oxford, UK), GLUT1 (ab14683, 1:2500; Abcam, Cambridge, UK), LDHA (NBP1-48336, 1:2000; Novus Biologicals, Cambridge, UK) or β-tubulin (clone TUB2.1, 1:2500; Sigma-Aldrich, Dorset, UK). The chemiluminescent signal was detected using a UVITEC Alliance Q9 gel doc system and associated image acquisition software.
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