Total protein was extracted and quanti ed using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scienti c, USA), separated by 10% SDS-PAGE, and transferred to polyvinylidene uoride (PVDF) membranes. After blocked with 5% BSA for 1 h, the membrane was incubated at 4°C overnight with primary antibodies as follows: DDIT4 (1:1,000, Proteintech), acetyl-Histone H4 (Lys8) (1:750, ZENBIO), acetyl-Histone H3 (Lys27) (1:750, ZENBIO), p-S6k1(Thr389/Thr412) (1:1,000, ZENBIO), S6k1 (1:1,000, ZENBIO), p-4EBP1(Thr46) (1:1,000, ZENBIO), 4EBP1 (1:1,000, ZENBIO), and β-actin (1:10,000, ZENBIO). After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, immunoreactive bands were detected by ECL Prime (GE Healthcare, UK) and an LAS-3000 imager (Fuji lm, Japan).
Ddit4
Manufactured by Proteintech
Sourced in China
DDIT4 is a gene that encodes a protein involved in the regulation of cellular processes. The protein plays a role in the cellular response to various stressors, including DNA damage and nutrient deprivation. The DDIT4 gene is commonly used in research applications to study these cellular stress response pathways.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Proteintech (2 mentions)
P p70s6k, by Cell Signaling Technology (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
β actin, by ZenBio (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Ki 67, by Solarbio (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
β tubulin, by Proteintech (1 mentions)
C myc, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Sds page protein loading buffer 5x, by Beyotime (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Cb 839, by Selleck Chemicals (1 mentions)
Alkbh5, by Abcam (1 mentions)
Compound 968, by Merck Group (1 mentions)
Ythdf2, by Proteintech (1 mentions)
Mettl14, by ABclonal (1 mentions)
6 protocols using ddit4
1
Western Blot Analysis of Protein Expression
2
Immunohistochemical Analysis of Tumor Tissues
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Surgically resected tumors and PDX tissues were formalin xed and embedded in para n, cut into 4 µm thick sections. H&E staining was used for assessment of pathology. For IHC, sections were treated with primary antibodies against human Ki-67 (1:100, Solarbio), P21 (1:100, Solarbio), DDIT4 (1:500, Proteintech). Next, they were incubated with secondary antibodies (Vectastain ABC Kit, Vector Laboratories, CA) at room temperature. Pathological examinations were performed under light microscopy by two pathologists blinded to the clinical information of patients.
3
Protein Expression Analysis with Immunoblotting
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Total protein was extracted with RIPA lysis buffer (Xianfeng Biotechnology, Xi'an, China), and quantitative analysis was conducted through the BCA Protein Assay Kit (Beyotime). Forty micrograms protein samples were loaded on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into polyvinylidene fluoride membrane (Millipore); then, they were sealed with 5% defatted milk for 1 h.
After sufficient cleaning, they were incubated with primary antibodies: DDIT4 (1:1000; Proteintech), P21 (1:2000; Proteintech), Bcl2 (1:2000; Proteintech), BAX (1:8000; Proteintech), cMYC (1:2000; Proteintech), CDK2 (1:2000; Proteintech), or β-tubulin (1:5000; Proteintech) at 4°C for 16 h. Subsequently, secondary antibodies (1:10,000; Proteintech) were incubated for 1 h. Protein signals were then visualized with ECL reagents (Beyotime).
After sufficient cleaning, they were incubated with primary antibodies: DDIT4 (1:1000; Proteintech), P21 (1:2000; Proteintech), Bcl2 (1:2000; Proteintech), BAX (1:8000; Proteintech), cMYC (1:2000; Proteintech), CDK2 (1:2000; Proteintech), or β-tubulin (1:5000; Proteintech) at 4°C for 16 h. Subsequently, secondary antibodies (1:10,000; Proteintech) were incubated for 1 h. Protein signals were then visualized with ECL reagents (Beyotime).
4
Protein Extraction and Western Blot Analysis
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To obtain total protein, cells were subjected to protein extraction using 1% PMSF and RIPA buffer (Solarbio, Beijing, China, Cat No. R0020) on ice for 30 minutes. The resulting mixture was centrifuged at 12,000 rpm for 30 minutes, and the protein suspension was collected from the liquid supernatant. Protein concentration was determined using the BCA method (Epizyme, Shanghai, China, Cat No. ZJ101). Subsequently, SDS-PAGE protein loading buffer (5X) (Beyotime, China) was added to the protein suspension, followed by boiling for 10 minutes. The protein was then separated using either a 10% or 12.5% SDS-PAGE gel (Epizyme, Shanghai, China, Cat No. PG113 or PG112) and transferred onto a 0.45 μm polyvinylidene fluoride (PVDF) membrane. To block the PVDF membranes, 5% skim milk was applied for 1.5 hours. Next, the membranes were incubated with primary antibodies including DDIT4 (ProteinTech, Wuhan, China, Cat No. 67059-1-Ig, 1:1000), BCL2 (ProteinTech, Wuhan, China, Cat No. 68103-1-Ig, 1:1000), Caspase-3 (Huaan, Hangzhou, China, Cat No. ET1602-39, 1:1000), and GAPDH (Huaan, Hangzhou, China, Cat No. ET1601-4, 1:5000), followed by incubation with corresponding secondary antibodies. Finally, the protein bands were visualized using chemiluminescence kits.
5
Molecular Mechanisms of mRNA Modification
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ATF4 (rabbit, 11815S), p-mTOR (rabbit, 5536S), mTOR (rabbit, 2983S), p-p70S6K (rabbit, 9205S), p70S6K (rabbit, 2708S), pS6 (rabbit, 4857S), LC3B (rabbit, 3868S), and sequestosome 1 (SQSTM1, rabbit, 8025) monoclonal antibodies were purchased from Cell Signaling Technology (Shanghai, China). DDIT4 (rabbit, 10638-1-AP) and YTHDF2 (rabbit, 24744-1-AP) polyclonal antibodies were purchased from Proteintech. FTO (rabbit, ab126605), WTAP (rabbit, ab195380), ALKBH5 (rabbit, ab69325), and m6A (mouse, ab208577) antibodies were purchased from Abcam. METTL3 (rabbit, a8370), and METTL14 (rabbit, a8530) polyclonal antibodies were purchased from ABclonal. Compound 968 was purchased from Sigma-Aldrich (SML1327). Actinomycin D was purchased from Selleck Chemicals (S8964). Chloroquine (CQ) was obtained from Sigma-Aldrich (C6628). Meclofenamate sodium was purchased from MCE (HY-B1320). CB839 was purchased from Selleck Chemicals (S7655).
6
Analysis of Kidney Graft Proteins
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Proteins from the grafted kidneys were extracted and then used for separation and transfer. The membranes were blocked and then incubated with primary antibodies against T‐bet, GATA3, Foxp3, RORγt (Abcam), DNMT1 (Novus Biologicals), p‐Akt, p‐mTOR, p‐P70S6K, PTEN (Cell Signaling Technology, Danvers, MA), DDIT4, and GAPDH (Proteintech, Rosemont, IL). After washing, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies and subsequently washed again. Chemiluminescence signals were detected after exposure to Immobilon Forte Western HRP substrate (EMD Millipore, Burlington, MA) using the ChemiDocTM XRS+System (Bio‐Rad, Hercules, CA).
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