5000 cells were seeded in laminin-coated 24-well plates. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.1% Triton X100 for 15 min at room temperature. The cells were then incubated overnight at 4 ℃ with CIC antibody (NB110-59,906, NOVUS). Samples were rinsed three times in PBS and incubated for 1 h at room temperature with AlexaFluor 594 donkey anti-rabbit secondary antibody (Thermo Fisher Scientific). Nuclei were stained with 0.5 µg/ml DAPI for 1 min at room temperature. Images were acquired using fluorescence microscopy. The primary antibodies used for the immunoblots: anti-CIC (NB110-59,906, NOVUS), anti-ETV4 (10,684–1-AP, Proteintech), anti-xCT (NB300-318, NOVUS), anti-ACTB (A3854, Sigma), anti-GAPDH (2118, Cell Signaling).
Nb300 318
Manufactured by Novus Biologicals
Sourced in United States
The NB300-318 is a laboratory product manufactured by Novus Biologicals. It is a piece of equipment designed for general laboratory use. No further details about its core function or intended use are available.
Automatically generated - may contain errors
Lab products found in correlation
Ab125066, by Abcam (4 mentions)
α tubulin, by Merck Group (2 mentions)
Glutathione assay kit, by Abcam (2 mentions)
Phospho eif2α, by Abcam (2 mentions)
Total eif2α, by Merck Group (2 mentions)
Hpa042309, by Atlas Antibodies (2 mentions)
Ab16667, by Abcam (2 mentions)
Mab3249, by R&D Systems (2 mentions)
Anti actb, by Merck Group (1 mentions)
Alexa fluor 594 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Nb110 59 906, by Novus Biologicals (1 mentions)
Total protein extraction kit, by BioChain (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
F1804, by Merck Group (1 mentions)
Ab92821, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Hpa007196, by Atlas Antibodies (1 mentions)
Bs 6313r, by Bioss Antibodies (1 mentions)
Ac026, by ABclonal (1 mentions)
15 protocols using nb300 318
1
Immunofluorescence and Immunoblot Analysis
2
Quantitative Western Blot Analysis of xCT
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Tissue samples were homogenized using a total protein extraction kit (BioChain) following the manufacturer’s instructions. Protein concentration in lysates was determined by bicinchoninic acid (BCA) protein assay (Pierce). Rabbit anti-xCT antibody (1:2000 dilution, Novus NB300-318) was used according to a standard western blotting protocol. After enhanced chemiluminescence (ECL) exposure, blots were washed and re-probed with rabbit anti-actin antibody (Cell Signaling Technology 4967S; 1:5000) as a loading control. To verify efficient extraction of xCT from the membrane during tissue lysis, an anti-Na,K-ATPase antibody (Cell Signaling Technology 3010S; 1:5000) was used as an additional control for western blotting [16 (link)]. Blots were scanned and signal was quantified using ImageJ (National Institutes of Health).
3
Immunoblot Analysis of Cystine Transporter Regulation
4
Cell Lysis and Protein Detection
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Whole-cell extracts were prepared from 1 × 106 cells using (RIPA) buffer (50 mM Tris-Cl, pH 6.8, 100 mM NaCl, 1%Triton-X-100, 0.1% SDS) supplemented with complete mini protease inhibitor cocktail (Roche Applied Sciences) for 20 minutes on ice and processed according to standard protocols. Anti-SLC7A11 antibody (Novus NB300-318) was used for detection with chemiluminescence.
5
Immunoblot Analysis of Cystine Transporter Regulation
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Immunoblots were prepared as previously described (41 (link)) and membranes were probed with antibodies against Slc7a11 (Novus Biologicals, NB300-318), UPF1/Rent1 (sc H-300), UPF2/Rent2 (kindly provided by J. Lykke-Andersen), Phospho-eIF2α (Epitomics, 1090-1), Total eIF2α (sc-11386), ATF4 (sc-200) and α-Tubulin (T9026, Sigma). RNA assessment: Isolation of RNA, cDNA generation, real-time PCR, DRB treatment and RNA stability experiment were done as described previously ((16 (link)). Real time primers for SLC7A11 are; human, 5′-GGGCATGTCTCTGACCATCT-3′ and 5′-TCCCAATTCAGCATAAGACAAA-3′; mouse 5′-TTGCAAGCTCACAGCAATTC-3′ and 5′-AGGGCAACCCCATTAGACTT-3′. Glutathione, cysteine transport, and cell viability assays: Intracellular glutathione was determined fluorometrically by using a Glutathione Assay Kit (Bio Vision, K264-100) and by liquid chromatography/Mass Spec (Metabolon, Durham, North Carolina) as previously described (17 ). Intracellular cysteine was also assessed by LC/MS by Metabolon.
6
Western Blot Analysis of m6A Regulators
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Western blotting was performed as previously reported 22 (link)-24 (link). Briefly, total proteins were extracted from human aorta samples or HASMCs by using radioimmunoprecipitation assay (RIPA) lysis buffer. The proteins were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. After that, the membrane was blocked with 5% nonfat milk for 90 min and then incubated with the indicated primary antibody overnight at 4 °C. The primary antibodies used in this study were as follows: ALKBH5 (HPA007196, Atlas Antibodies), AIFM2/FSP1 (HPA042309, Atlas Antibodies), METTL14 (HPA038002, Atlas Antibodies), METTL3 (15073-1-AP, Proteintech), WTAP (60188-1-Ig, Proteintech), SLC3A2 (15193-1-AP, Proteintech), FTO (ab92821, Abcam), GPX4 (ab125066, Abcam), β-Actin (#8457, Cell Signaling Technology), SLC7A11 (NB300-318, Novus Biologicals), and Flag (F1804, Sigma-Aldrich) antibodies.
7
Comprehensive Antibody Panel for Oxidative Stress Analysis
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The following primary antibodies were used for immunoblot analysis and immunohistochemistry: mouse monoclonal anti–transferrin receptor antibody (13‐6800, Invitrogen), goat polyclonal anti–Lipocalin‐2/NGAL antibody (AF1857, R&D systems), rabbit monoclonal anti–β‐actin (ACTB) antibody (AC026, ABclonal), rabbit monoclonal anti–IRP1 antibody (ab126595, Abcam), rabbit polyclonal anti–IRP2 antibody (NB100‐1798, Novus), rabbit polyclonal anti–FTH1 antibody (3998, CST), rabbit polyclonal anti–ferritin light chain (FTL) antibody (ab69090, Abcam), rabbit polyclonal anti–divalent metal transporter 1 (DMT1) antibody (20507‐1‐AP, proteintech), rabbit polyclonal anti–8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG) antibody (bs1278R, Bioss), rabbit polyclonal anti–4‐Hydroxynonenal antibody (bs6313R, Bioss), anti–CD10 antibody (ab256494, Abcam), anti–Ki67 antibody (ab16667, Abcam), rabbit monoclonal anti–Cleaved Caspase‐3 antibody (9664, CST), rabbit monoclonal anti–Glutathione Peroxidase 4 antibody (ab125066, Abcam), and anti–xCT antibody (NB300‐318, Novus). Secondary antibodies used in immunoblot analysis and immunohistochemistry were as follows: HRP‐conjugated polyclonal Goat anti–Rabbit antibody (P0448, DAKO), HRP‐conjugated polyclonal Rabbit anti–mouse antibody (P0260, DAKO) and HRP‐conjugated polyclonal Rabbit anti–Goat antibody (P0160, DAKO).
8
Immunofluorescence Staining of XCT Protein
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66.1 cells were seeded on gelatin-coated glass coverslips, fixed at 48 hours with ice-cold 70% ethanol and permeabilized with 0.25% Triton. Coverslips were incubated with anti-XCT polyclonal antibody (NB300-318; Novus Biologicals, Littleton, CO; 1:50 dilution in 5% BSA). AlexaFluor 488 goat anti-rabbit secondary antibody (2.5 μg/mL; Life Technologies, Carlsbad, CA) was prepared in 5% BSA with 1% normal donkey serum. Coverslips were mounted on glass slides with ProLong Gold antifade reagent with DAPI (Molecular Probes, Eugene, OR). Slides were imaged on a Zeiss Axioskop 40 using a 63x/0.08 numerical aperture Achroplan objective. Images were captured with a Zeiss AxioCam-Cm 1.
9
Immunoblot and Immunohistochemistry Analysis
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Antibodies to HMGA1 (1:1000, sc-393213, Santa Cruz), SLC7A11 (1:1000, #12691, Cell Signaling), cleaved caspase-3 (Asp175, 1:1000, #9661, Cell Signaling), ATF4 (1:1000, #11815, Cell Signaling), DDDDK tag (1:1500, ab205606, Abcam), and β-actin (1:20000, #66009-1-Ig, Proteintech) were used for the immunoblot. HMGA1 (1:1000, sc-393213, Santa Cruz), xCT (1:200, NB300-318, NOVUS), Ki67 (1:200, ab16667, Abcam), and 4-hydroxynonenal (1:5000, MAB3249, R&D systems) were used for the immunohistochemistry. Ferrostatin-1 (HY-100579), erastin (HY-15763), staurosporine (HY-15141), z-VAD-FMK (HY-16658B), necrostatin-1 (HY-14622A), and cisplatin (HY-17394) were purchased from MCE (MCE, China).
10
Quantifying Cisplatin Resistance Factors
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