Following drug treatment, cells were washed with PBS and mixed with lysis buffer. The mixtures were vortexed for 1 min and placed on ice for 30 min. Following centrifugation (10,000 × g for 10 min, 4°C), the dye-binding Bradford method (Bio-Rad Laboratories, Inc.) was used to quantify the proteins. Equal amounts of protein (40 µg protein/lane) were separated on a 10–12% SDS gel via PAGE and transferred onto PVDF membranes. The membranes were separately incubated with the primary anti-DKK4 rabbit monoclonal IgG antibody, anti-transcription factor AP-2 epsilon (TFAP2E) rabbit monoclonal IgG antibody (AV40023-100UG, 1:1,000 dilution; Sigma-Aldrich; Merck Millipore), anti-hypoxia-inducible factor-2α (HIF2α) rabbit polyclonal IgG antibody (ab109616, 1:1,000 dilution; Abcam) and anti-GAPDH rabbit monoclonal IgG antibody (ab9483, 1:1,000 dilution; Abcam), overnight at 4°C. The secondary antibody was a horseradish peroxidase-labeled goat anti-rabbit IgG antibody (H+L). The samples were incubated with the secondary antibody for 1 h at 37°C. The signals were analyzed following treatment with TMB substrate and visualized by the ChemiDocä Touch Imaging system.
Ab109616
Manufactured by Abcam
Sourced in United States
Ab109616 is a monoclonal antibody that recognizes the human protein GAPDH. GAPDH is a key enzyme involved in glycolysis, a fundamental metabolic process. This antibody can be used for the detection of GAPDH in various applications such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab9483, by Abcam (1 mentions)
Bradford dye binding method, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab9485, by Abcam (1 mentions)
Ecl kit, by Aspen (1 mentions)
Ab226977, by Abcam (1 mentions)
Ab228649, by Abcam (1 mentions)
Ab137704, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Sc 8432, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Ab108981, by Abcam (1 mentions)
Ab51608, by Abcam (1 mentions)
Inform software, by PerkinElmer (1 mentions)
Vectra slide scanner, by PerkinElmer (1 mentions)
Ab175471, by Abcam (1 mentions)
9 protocols using ab109616
1
Western Blot Analysis of Protein Targets
2
Western Blot Analysis of EPAS1 Protein
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Protein samples were electrophoresed in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels, and resolved proteins were electro‐transferred onto polyvinylidene difluoride membranes (Millipore) in a transferring buffer (25 mM Tris, 0.2 M glycine and 25% methanol). After blocking with 5% skimmed milk, the membranes were incubated with anti‐EPAS1 (Abcam, Ab109616, 1:1000 dilution) and anti‐GAPDH antibodies (Abcam, Ab9485, 1:2500 dilution), followed by incubation with appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies (Abcam, Ab205718, 1:30000 dilution). An enhanced chemiluminescence ECL kit (ASPEN) was used to visualize the bands.
3
Protein Extraction and Western Blot Analysis
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Total proteins were isolated from cells with RIPA buffer (Beyotime, Hangzhou, China). The 10% SDS-PAGE gels separated protein, then transferred the protein to PVDF membranes (Millipore, Billerica, MA, USA). After being blocked by 5% nonfat milk for 2h, antibodies for HDAC3 (1:1000, ab137704, Abcam, USA), and HIF-1α (1:1000, ab228649, Abcam, USA), HIF-2α (1:1000, ab109616, Abcam, USA), BCL3 (1:1000, ab216877, Abcam, USA), CCND1 (1:1000, ab226977, Abcam, USA), β-actin (1:1000, sc-8432, Santa Cruz, USA) were used to incubate membranes at 4 °C overnight. Then, the membranes were incubated by the HRP-conjugated secondary antibodies (1:1000, Proteintech, USA) for 1-2h. The blots were detected using an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).
4
Immunohistochemistry of HIF-2α in Tissue
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Immunohistochemistry detected cells stained by HIF-2α primary antibody (Abcam [ab109616], 1:500 ab:PBST) within formalin-fixed, decalcified, paraffin-embedded sections. Details in e-methods (links.lww.com/NXG/A252 ).
5
Correlation of HIF and SENP1 in RCC
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To investigate the correlation of expression of HIF’s and SENP1 genes, the publicly available Human Protein Atlas database of RCC samples (N = 877) were analyzed for the correlation (R^2) or covariation by Spearman correlation methods for RNA expression of HIF1α, HIF2α, and SENP1 genes. For tissue microarray (TMA) analysis, malignant and tumor-adjacent benign tissues were used to construct a manual tissue array [43 (link)]. A total of 471 malignant cores and 190 benign cores were included. Immunohistochemistry was performed by UW Translational Research Initiatives in Pathology (TRIP) facility. TMA with human samples was performed the protocol (#2011-0179) approved by IRB. Anti-SENP1 (ab108981), anti-HIF1α (ab51608), or anti-HIF2α (ab109616), all from Abcam, MA, was applied to the TMAs, and hematoxylin was used for count-staining. Stained slides were scanned by Vectra slide scanner (PerkinElmer, MA) and SENP1, HIF1α, and HIF2α expression levels in the nuclears (where SENP1 would act on these HIF proteins) were quantified and analyzed in inForm software (PerkinElmer).
6
Immunofluorescence Staining of Cellular Markers
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For immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 10 min, incubated in blocking solution (5% goat serum and 0.5% Triton X-100 in PBS) for 30 min and then incubated with unconjugated anti-α-SMA antibody (A5228; Sigma-Aldrich, St Louis, MO, USA), anti-HIF-2α antibody (ab109616; Abcam or NB100-132; Novus) or anti-CD31 antibody (ab28364; Abcam). Cells were then incubated with Alexa-Fluor 488 (green) conjugated anti-mouse IgG2a antibody (Invitrogen, Carlsbad, CA, USA), Alexa-Fluor 568 (red) conjugated anti-mouse IgG antibody (ab1500113; Abcam), or Alexa-Fluor 568 (red) conjugated anti-rabbit IgG antibody (ab175471; Abcam) and with Hoechst 33342 (Sigma-Aldrich) for 60 min. Cells were imaged and images captured with an IX83 microscope (Olympus, Tokyo, Japan) and the ratios of α-SMA- or HIF-2α-positive cells to total cell numbers estimated from Hoechst 33342 nuclear staining were calculated using Image-J (National Institutes of Health, Bethesda, MD, USA).
7
Western Blot Analysis of HIF-1α and HIF-2α
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Cells were lysed in RIPA Buffer (Nacalai Tesque, Inc., Kyoto, Japan) containing a protease and phosphatase inhibitor cocktail (Fujifilm Wako Pure Chemical Corporation, Kyoto, Japan). Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Ten micrograms of protein were denatured in Laemmli sample buffer and loaded on 4–15% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Richmond, CA, USA). After electrophoretic separation, the proteins were transferred onto PVDF membranes (MilliporeSigma, Billerica, MA, USA). Membranes were blocked overnight at 4 °C with Tris-buffered saline (TBS) containing 5% non-fat dried milk or bovine serum albumin. Following blocking, membranes were incubated overnight at 4 °C with primary antibodies, including anti-HIF-1α (ab179483; 1:1000; Abcam, Cambridge, UK), anti-HIF-2α (ab109616; 1:1000; Abcam), and anti-β-actin (GTX109639; 1:1000; GeneTex, Irvine, CA, USA). Proteins were incubated with secondary antibodies (anti-rabbit or anti-mouse IgG antibody; Cat. 7074 and Cat. 7076, respectively; 1:20,000; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in TBS containing 5% non-fat dried milk. The chemiluminescence signal was detected using enhanced chemiluminescent substrate (SuperSignal™ West Femto Trial Kit; Thermo Fisher Scientific).
8
Quantifying Cell Migration via Wound Scratch Assays
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Wound scratch assays were performed as described elsewhere (Loriot et al., 2015) and analyzed using ImageJ software or using Nikon videomicroscope and NIS-advanced imaging software. The experiments were performed in triplicates.
shRNA stable transduction Lentiviral constructs were purchased at Sigma-Aldrich. pLKO.1 is the parental viral vector without shRNA. WT imCC (30000) were infected in a serum-free medium containing 8 mg/mL polybrene by either 10 MOI of Tet1 (TRCN0000341849) and 10 MOI of Tet2 anti-TET2 (Proteintech #21207-1-AP 1/400), anti-TET3 (Active motif #61744 clone 23B9 1/400), anti-HIF2a (Abcam #ab8365 1/1000; Figure 6) or, anti-HIF2a (Abcam ab109616, 1/500; Figure 7).
For H3K27me3 and H3-Cterminal western blots, proteins were extracted by acidic lysis with 0,2M H2SO4, and precipitated with trichloroacetic acid. Pellets were washed with acetone and resuspended in diluted loading buffer and sonicated. Western blot conditions were identical. Proteins were transferred on nitrocellulose membrane. Anti-H3K27me3 (Active Motif #39156) was diluted 1/1000 and anti-H3-Cterminal (Active Motif #39164) was diluted 1/2000.
shRNA stable transduction Lentiviral constructs were purchased at Sigma-Aldrich. pLKO.1 is the parental viral vector without shRNA. WT imCC (30000) were infected in a serum-free medium containing 8 mg/mL polybrene by either 10 MOI of Tet1 (TRCN0000341849) and 10 MOI of Tet2 anti-TET2 (Proteintech #21207-1-AP 1/400), anti-TET3 (Active motif #61744 clone 23B9 1/400), anti-HIF2a (Abcam #ab8365 1/1000; Figure 6) or, anti-HIF2a (Abcam ab109616, 1/500; Figure 7).
For H3K27me3 and H3-Cterminal western blots, proteins were extracted by acidic lysis with 0,2M H2SO4, and precipitated with trichloroacetic acid. Pellets were washed with acetone and resuspended in diluted loading buffer and sonicated. Western blot conditions were identical. Proteins were transferred on nitrocellulose membrane. Anti-H3K27me3 (Active Motif #39156) was diluted 1/1000 and anti-H3-Cterminal (Active Motif #39164) was diluted 1/2000.
9
Western Blot Analysis of Nasal Mucosa Proteins
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Total protein was extracted by adding radioimmunoprecipitation assay lysis buffer (Solarbio) to mouse nasal mucosa tissues, and protein concentration was measured using a bicinchoninic acid kit (Sigma). Samples containing 20 μg of protein were boiled, and the proteins were separated by 10% polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene fluoride membranes (Millipore). The membrane was sealed in 5% skim milk (BD, San Jose, CA, USA) for 1 h at room temperature and then incubated with primary antibodies to XBP1 (1 : 1000, ab220783, Abcam), HIF-1a (1 : 2000, GTX127309, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), HIF-2a (1 : 1000, ab109616, Abcam), HIF-3a (1 : 1000, GTX85702, GeneTex), β-catenin (1 : 1000, ab223075, Abcam), and GAPDH (1 : 10,000, ab181603, Abcam) overnight at 4°C. The membranes were then incubated with goat anti-rabbit secondary antibody (1 : 5000, ab205718, Abcam) for 60 min at 37°C and developed by exposure to an enhanced chemiluminescence substrate kit (Abcam). Gray value analysis of the target gene was performed by Image J (National Institutes of Health, Bethesda, MD, USA) with GAPDH as the internal reference gene.
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