Cell line and tumor total RNA was extracted using RNAzol (Sigma) according to the manufacturer’s protocol. RNA (1ug) was used to generate cDNA with the High-Capacity cDNA Reverse Transcription Kit (Thermo). For qPCR, we used the StepOnePlus instrument (Thermo) with pre-designed TaqMan gene expression assays (Thermo): CXCR4 (Hs00607978_s1), DARS (Hs00154683_m1), IL6 (Hs00174131_m1), IL8 (Hs00174103_m1) and TBP (Hs00427620_m1). TBP served as housekeeping control and data was analyzed using the double delta Ct method. For immunoblotting sub-confluent cells were pelleted, washed with cold PBS and lysed with RIPA buffer. Nuclear and cytoplasmic extracts were obtained using the NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo) according to the recommendations of the manufacturer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes and blotted with antibodies against CXCR4 (Abcam, ab124824, 1:500), HIF2α (Novus, NB100-122, 1:500), VHL (Cell Signaling, 2738, 1:500), phospho-p65 (Cell Signaling, 3033, 1:1000), p65 (Santa Cruz, sc8008, 1:1000), TBP (Abcam, ab63766, 1:5000), B-tubulin (Abcam, ab6046, 1:10000) and ß-actin (Sigma, A5441, 1:5000), ß-tubulin (Sigma, T5201, 1:5000). Secondary antibodies were HRP-conjugated (Dako, 1:5000).
Nb100 122
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
NB100-122 is a laboratory reagent produced by Cell Signaling Technology. It is an antibody that is designed for use in various experimental techniques, such as immunohistochemistry and western blotting, to detect the presence and expression levels of a specific target protein.
Automatically generated - may contain errors
Lab products found in correlation
Secondary anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Nb100 479, by Cell Signaling Technology (2 mentions)
Secondary anti mouse igg hrp, by Cell Signaling Technology (2 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Rnazol, by Merck Group (1 mentions)
A5441, by Merck Group (1 mentions)
Ab6046, by Merck Group (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Ab63766, by Abcam (1 mentions)
Sc 8008, by Abcam (1 mentions)
T5201, by Merck Group (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Wbkls0100, by Merck Group (1 mentions)
Nb600 408, by Abcam (1 mentions)
Ab7817, by Cell Signaling Technology (1 mentions)
Bca protein kit, by Merck Group (1 mentions)
Hif 1a, by Novus Biologicals (1 mentions)
Mab374, by Merck Group (1 mentions)
4 protocols using nb100 122
1
Comprehensive RNA and Protein Analysis Pipeline
2
Western Blot Analysis of Kidney Samples
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The procedures for western blot analysis were described elsewhere44 (link). Kidney tissues and cells were subjected to western blot analysis using standard procedure. After blocking nonspecific binding with 5% nonfat milk in phosphate-buffered saline solution (PBST) for 1 h at room temperature, cell membranes were incubated with primary antibody overnight at 4 °C. Then, they were immunoblotted with antibodies against SIRT1 (Abcam; ab18239), fibronectin (Sigma; CP70), collagen I (Novus; NB600-408), α-SMA (Abcam; ab7817), HIF-1α (Cell Signaling Technology; #36169), HIF-2α (Novus; NB100-122), Ac-K (Cell Signaling Technology; #9441), and GAPDH (Abcam; ab8245), as well as the appropriate secondary antibodies. Band intensity was captured using a chemiluminescence reagent (WBKLS0100, Millipore) with a GE ImageQuant LAS 4000. Image J software was used to analyze the densitometry of the bands.
3
Western Blot Analysis of Epigenetic Regulators
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Following passage, cells were first washed with PBS before being lysed with fresh RIPA buffer and centrifuged at 10,000× g for 10 min at 4 °C. Total protein concentration in the supernatant was measured using the BCA protein kit (Sigma-Aldrich). Western Blot analysis was performed on 30 μg of protein using DNMT3B (R&D System/MAB7646, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK, TET1 (ThermoFisher/GT1462, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK, HIF1A (Novusbio/NB100-479, Secondary Anti-rabbit IgG-HRP Cell Signalling/70745, London, UK, HIF2A (Novusbio/NB100-122, Secondary Anti-rabbit IgG-HRP Cell Signalling/70745), and GAPDH (Merck/MAB374, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK). The immunoreactivity bands were subjected to UpLight HRP chemiluminescent substrate solution (Uptima) and imaged using a FluorChem M Imager.
4
Epigenetic Regulators Expression Analysis
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BCA protein assay (Sigma-Aldrich) was performed to measure protein concentrations. 30μg of total protein was used for western blot analysis using antibodies against DNMT3B (R&D System/ MAB7646, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765), TET1 (ThermoFisher/ GT1462, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK), HIF1A (Novusbio/NB100-479, Secondary Anti-rabbit IgG-HRP Cell Signalling/70745, London, UK), HIF2A (Novusbio/NB100-122, Secondary Anti-rabbit IgG-HRP Cell Signalling/70745, London, UK) and GAPDH (Merck/MAB374, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK). Blots were imaged using a FluorChem M Imager system.
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