Journal: Journal of translational medicine

Article Title: Establishment of a prognostic model based on ER stress-related cell death genes and proposing a novel combination therapy in acute myeloid leukemia.

doi: 10.1186/s12967-025-06615-y

Figure Lengend Snippet: Fig. 9 DDIT4 promotes AML cell proliferation in vitro. A Knockdown of DDIT4 in U937 and HL60 cell lines. B‒H Analysis of cell viability (B), cell counting (C), colony formation (D‒E), cell apoptosis (F), the cell cycle (G), and migration (H) in control and DDIT4-knockdown cells. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: The primary antibodies used for the western blotting assays were rabbit antibodies against DDIT4 (1:1000, 10638-1- AP; Proteintech, Wuhan, Beijing) and mouse antibodies against beta-actin (1:1000, AC004; ABclonal Technology, Wuhan, China).

Techniques: In Vitro, Knockdown, Cell Counting, Migration, Control

Journal: Frontiers in Bioscience-Landmark

Article Title: The Role of CEBPD in Oxidative Stress and Angiogenesis Regulation in Endometriosis

doi: 10.31083/fbl33488

Figure Lengend Snippet: Fig. 4. CEBPD promotes DDIT4 expression in 12Z cells. (A) Differential expression volcano map. Blue dots indicate significantly

Article Snippet: Immunohistochemical staining was performed on the tissue sections using anti-human CEBPD (1:200, #ab245214, Abcam, Cambridge, MA, USA) and anti-human DDIT4 (1:100, #10638-1-AP, Proteintech, Chicago, IL, USA).

Techniques: Expressing, Quantitative Proteomics

Journal: Frontiers in Bioscience-Landmark

Article Title: The Role of CEBPD in Oxidative Stress and Angiogenesis Regulation in Endometriosis

doi: 10.31083/fbl33488

Figure Lengend Snippet: Fig. 5. CEBPD promotes oxidative stress and proliferation by targeting DDIT4 in 12Z cells. (A) The expression levels of CEBPD and DDIT4 in different groups were analyzed by Western blot (the p-values for CEBPD and DDIT4 in other groups: sh-control group

Article Snippet: Immunohistochemical staining was performed on the tissue sections using anti-human CEBPD (1:200, #ab245214, Abcam, Cambridge, MA, USA) and anti-human DDIT4 (1:100, #10638-1-AP, Proteintech, Chicago, IL, USA).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Bioscience-Landmark

Article Title: The Role of CEBPD in Oxidative Stress and Angiogenesis Regulation in Endometriosis

doi: 10.31083/fbl33488

Figure Lengend Snippet: Fig. 6. CEBPD activates the ERK pathway in 12Z cells through DDIT4. (A) The expression of CEBPD in different groups was

Article Snippet: Immunohistochemical staining was performed on the tissue sections using anti-human CEBPD (1:200, #ab245214, Abcam, Cambridge, MA, USA) and anti-human DDIT4 (1:100, #10638-1-AP, Proteintech, Chicago, IL, USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: A) Heatmap view of average differences in allelic fractions (AF) in the comparison between polysome-associated and total RNA for the 40 tranSNPs identified from the RNA-seq data from either mock or Nutlin-treatment conditions (Table S1 and S2 present the AF value for each biological replicate; accession number for the RNA-seq data: GSE95024). AF was computed as the number of reads for the alternative allele over the sum of reads of alternative and reference alleles. B ) Plots of the AF for the rs1053639 -in DDIT4-, rs763121 -in DDX17-, and rs6588 -in PERP-tranSNPs obtained through RNA-seq of cytoplasmic total (TOT) and polysome bound mRNAs (POL). Individual replicates and the AF mean are plotted. *p-value < 0.05, two-tailed, paired t-test. C) AF values of the selected tranSNPs calculated from Sanger sequencing electropherograms, comprising also the sub-polysomal-RNP to 80S-(SUB) fractions. Individual replicates and the AF mean are plotted. *p-value < 0.05, two-tailed, paired t-test. D) Results of the dual luciferase assays presenting the relative activity (average and individual biological replicates) for the reference (REF_) and alternative (ALT_) alleles for each of the three selected tranSNPs cloned separately into the pGL4.13 vector immediately downstream the Firefly luciferase stop codon. Data was normalized using Renilla luciferase. **p-value < 0.01. ***p-value < 0.0005. ****p-Value < 0.0001, two-tailed, unpaired t-test.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Comparison, RNA Sequencing, Two Tailed Test, Sequencing, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: Parental HCT116 cells - heterozygous for the rs1053639 SNP - were edited to obtain homozygous clones for the A and the T alleles. A) CRISPR/Cas9-mediated knock-in was employed for this aim. We annealed a gene-specific crRNA with a tracrRNA, obtaining a sgRNA for DDIT4. The same sgRNA cut both alleles since the SNP represented the “N” base of the-NGG PAM. An 85-nt long donor DNA with the endogenous sequence (mixture of 50% A and 50% T) was purchased to be used as a template. B) Representative electropherograms of Sanger sequencing analyses indicating the genotype of the selected clones for rs1053639.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Clone Assay, CRISPR, Knock-In, Sequencing

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: HCT116 cells were edited by CRISPR/Cas9 RNPs and donor DNA to obtain cell trios with the three genotypes for the rs1053639 tranSNP located in the proximal portion of the DDIT4 3’UTR. A) Western blot analysis of DDIT4 protein expression in HCT116 cells (A/T heterozygous) and DDIT4 rs1053639 homozygous clones (TT, AA). β-Tubulin was used as a reference. B) Densitometry analysis of the blots from several biological replicates shows a significant difference in relative DDIT4 protein abundance with the different tranSNPs genotypes. ***P-value <0.005 Ordinary one-way ANOVA. C) Western blot analysis of DDIT4 protein in HCT116 TT and AA homozygous clones for the rs1053639 treated with 100µg/ml cycloheximide (CHX) at the indicated time points. β-Tubulin was used as a reference. D) (Left) Relative DDIT4 mRNA expression as measured by RT-qPCR and normalized to GAPDH and YWHAZ. Student’s t-test shows no significant differences in DDIT4 mRNA expression in the different clones. ns, not significant. (Right) Estimation of DDIT4 mRNA stability following inhibition of transcription by actinomycin D treatment (10µg/ml). The curve presents the residual relative DDIT4 mRNA levels (average and standard deviation of at least three biological replicates) at various treatment time points measured by RT-qPCR decay from HCT116 clones harboring TT or AA genotypes. The estimated half-lives of the DDIT4 mRNA in TT and AA homozygous clones are indicated. E) DDIT4-AS1 and DDIT4 mRNA levels measured by RT-qPCR and normalized to GAPDH across TT and AA clones. The expression of DDIT4-AS1 was neglectable compared to the expression of the sense DDIT4 mRNA in our cellular models. Data represents the mean ± SD of three independent experiments. ns, not significant. F) HCT116 clones were exposed to 100nM thapsigargin (Tg) or vehicle for 4 hours, and the induction of DDIT4 mRNA was measured by RT-qPCR with clones of the three rs1053639 genotypes. Average, standard deviation, and individual replicates are presented. ns, not significant, student’s t-test. G-H ) Western blot and densitometry analysis showing relative DDIT4 protein induction upon thapsigargin (Tg 100nM) treatment of the three rs1053639 genotypes. β-Tubulin served as a control. ***p-value <0.005, unpaired t-test.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: CRISPR, Western Blot, Expressing, Clone Assay, Quantitative RT-PCR, Inhibition, Standard Deviation, Control

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: HCT116 parental cells and clones differing for the rs1053639 homozygous genotypes were treated with either Nutlin 10 µM or vehicle control for 16 hours. A) Cell lysates were analyzed by immunoblotting for the induction of p53 and DDIT4 protein levels and the phosphorylation state and levels of the eIF4EBP1 mTORC1 target. β-Tubulin and GAPDH were used as references. B, C) Densitometry analysis of p53 and DDIT4 protein levels shows significant induction upon Nutlin treatment. D) Densitometry analysis of phosphorylated eIF4EBP1 blots reveals that significant inhibition of the phosphorylation of eIF4EBP1 is apparent only in the rs1053639 TT homozygous clones. ***p-value <0.005. **p-value <0.001. *p-value <0.05. ns, not significant. unpaired t-test.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Clone Assay, Control, Western Blot, Inhibition

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: Cytoplasmic lysates from HCT116 rs1053639 homozygous (TT, AA) clones treated with thapsigargin 100nM or vehicle for four hours, were subjected to fractionation through sucrose density gradients (15 to 50%). Polysome profiles are shown in . A-D) The distribution of DDIT4 mRNA (Mock, thapsigargin) across the 12 gradient fractions was quantitatively analyzed across the two genotypes by RT-qPCR. Relative mRNA % in every fraction was plotted. Three independent biological experiments were performed. CDKN1 B-E) and RPS26 C-F) mRNAs were used as control transcripts and were analyzed using the same methodology as described for DDIT4 mRNA.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Clone Assay, Fractionation, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: A) Structural RNA probes were designed based on RNA folding prediction tools. RNA EMSA was performed using the full-length recombinant RBMX protein and the above-mentioned RNA probes. In the ‘0’ lane, the RNA probe was incubated with the binding buffer and the protein elution buffer. RNA-protein complexes were resolved on a native 6% gel. B) The endogenous RBMX protein was immunoprecipitated from a whole cell lysate in TT and AA clones. Results are expressed as the percentage of the target DDIT4 mRNA relative to the input sample and normalized to the positive control CSDE1 mRNA to account for the variability in RIP efficiency. At least two independent biological replicates were performed. C) Cell fractionation was performed in HCT116 cells and protein levels were evaluated. Lamin A/C (70-75 kDa) was used as a marker of the nuclear fraction, and β-Tubulin (55 kDa) was used as a marker of the cytoplasmic fraction. DDIT4 protein was found exclusively in the cytoplasm. RBMX is mostly a nuclear protein as previously reported. WCL: whole cell lysate. CYTO: cytoplasm. D) RNA levels were evaluated in TT and AA cells upon cell fractionation followed by RT-qPCR. The distribution of DDIT4 mRNA is significantly different among TT and AA cells, with DDIT4 mRNA from TT cells being considerably more enriched in the cytoplasm. Data represent the mean ± SD of at least four independent experiments. The relative expression of the gene in the cytoplasm is calculated as the fold change of the Ct in the cytoplasm compared to the Ct in the nucleus, with nuclear and cytoplasmic RNAs eluted in the same volume of water. ****p-value < 0.0001. Unpaired t-test. E) U6 snRNA and NEAT1 were used as markers of the nuclear fraction, 7SL and GAPDH were used as markers of the cytoplasmic fraction. However, GAPDH mRNA is not absolutely cytoplasmatic, consistent with previous reports. Markers for the AA cells are shown on the left , and markers for the TT cells are shown on the right . F) RBMX was knocked down in TT and AA cells. DDIT4 mRNA levels in control (siNT) and knocked-down samples (siRBMX) were evaluated in TT and AA cells upon cell fractionation followed by RT-qPCR. Upon RBMX silencing, the intracellular distribution of DDIT4 mRNAs was slightly mitigated. Data represent the mean ± SD of three independent experiments. *p-value 0.0114 **p-value 0.0031, unpaired t-test. G) DDIT4 protein levels in control (siNT) and knocked-down samples (siRBMX) were evaluated in TT and AA cells upon cell fractionation followed by western blot. Representative blot of DDIT4 protein levels across TT and AA clones (Top). Densitometry analysis of the blots from several biological replicates shows that DDIT4 protein levels in TT clones decrease to the levels of the AA clones upon RBMX knock-down. *p-value < 0.05. Unpaired t-test. H) Confocal images of smiFISH-IF of cell clone #3 (TT) and clone #6 (AA) after control (siNT) or RBMX silencing for 72 hours. RBMX protein is shown in red, DDIT4 mRNA is in yellow. Scale bar is 10 μm. I) At least three fields (15-20 cells per field) for each coverslip and each condition were imaged, and cytoplasmic spots were quantified using Cell-Profiler 4.0.7 (Broad Institute, Inc.) and segmenting them as objects with a typical diameter range of 10 to 30 pixels. (Left panel), number of DDIT4 mRNA spots per cell. (Right panel), spot mean intensity. ** p< 0.01; *** p<0.001; **** p<0.0001, ordinary one-way ANOVA. ns = not significant.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Recombinant, Incubation, Binding Assay, Immunoprecipitation, Clone Assay, Positive Control, Cell Fractionation, Marker, Quantitative RT-PCR, Expressing, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: HCT116 parental cells and clones differing for the rs1053639 homozygous genotypes were treated with either thapsigargin 100nM or vehicle control for 4 hours. A, C) Cell lysates were analyzed by immunoblotting for the phosphorylation state and levels of the mTORC1 target eIF4EBP1 and S6K proteins. B, D) Densitometry analysis of all blots reveals that significant inhibition of the phosphorylation of both eIF4EBP1 and S6K proteins is apparent only in the rs1053639 TT homozygous clones. *p-value <0.05. ns, not significant. (E, F) Markers of the ER stress response are activated in HCT116 cells in response to thapsigargin treatment independently from the rs1053639 genotype. E) Western blot analysis of ATF4 protein induction upon thapsigargin (Tg 100nM) treatment in HCT116 cells (A/T heterozygous) and DDIT4 rs1053639 homozygous clones (TT, AA). β-Tubulin was used as a reference. F) Relative CHOP, SENS2, and spliced XBP1/ un-spliced XBP1 mRNA expression as measured by RT-qPCR and normalized to GAPDH and YWHAZ.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Clone Assay, Control, Western Blot, Inhibition, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: A) DDIT4 clones harboring the TT genotype were transduced with Lentiviral particles to constitutively express EGFP. After transduction, cells were treated with 5 µg/ml blasticidin for at least 15 days, to reach at least 95% of GFP+ cell populations. DDIT4 clones harboring the AA genotype (not fluorescent) were mixed ∼1:1 with EGFP-expressing TT genotype cells and co-cultured over 15 days. The relative growth was assessed by flow cytometry analyzing 10K cells. Cellular fitness of the GFP+ clones relative to GFP-competitor clones was calculated as follows: pGFP+(t) = NGFP+(t) / (NGFP+(t) + N GFP-(t)), W = ln ((pGFP+(ti) /pGFP-(ti)) / (pGFP+(t0) /pGFP-(t0)), adapting a described method . The error bars represent the mean ± SD of at least eight independent experiments (Four TT GFP+, four AA GFP+). **P-value <0.005. *P-value < 0.05 Ordinary two-way ANOVA. B) Cell cycle analysis by flow cytometry (FACS) for single clones before co-culture (Left) and sorted clones seven days after co-culture (Right). C) TT and AA clones were co-cultured as described in A) for seven days, treated with the pan-caspase inhibitor Q-VD-OPh 10µM or vehicle control, and analyzed by FACS at day 0, 3, and 7. The graph represents the proportion of GFP+ cells across time points and conditions. Doxorubicin 3µM was used as a positive control to induce apoptosis .

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Clone Assay, Transduction, Expressing, Cell Culture, Flow Cytometry, Cell Cycle Assay, Co-Culture Assay, Control, Positive Control

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: A) (Top) DDIT4 clones harboring the TT and AA genotypes were both separately transduced with Lentiviral particles to constitutively express either EGFP or MiRFP. After transduction, cells were treated with 5 µg/ml blasticidin for at least 15 days, to reach at least 95% of GFP+ or RFP+ cell populations. (Bottom) TT and AA clones were co-cultured using both combinations for the fluorophore. The graphs are representative of at least four replicates for each combination and show that the results were not influenced by the presence of the fluorescent marker. B) HCT116 clones were treated with Doxorubicin 3µM to induce apoptosis prior to Q-VD-OPh as a positive control (Left). TT and AA in co-culture were treated with Doxorubicin and /or Q-VD-OPh. The graph represents the proportion of the GFP+ cells after three days acquired by FACS.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Clone Assay, Transduction, Cell Culture, Marker, Positive Control, Co-Culture Assay

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: DDIT4 rs1053639 homozygous clones, transduced with lentiviral particles to constitutively express either EGFP or MiRFP as described in , were xenotransplanted into zebrafish larvae two days post-fertilization. Tumor growth was monitored over a three-day period, with a minimum of 30 larvae per genotype condition. Representative confocal microscopy images were captured on day 1 and day 3 post-transplantation, with TT clones shown in green and AA in purple. A) DDIT4 clones expressing GFP (TT clones) were mixed∼ 1:1 with RFP-expressing clones (AA genotype) and were initially co-cultured in vitro for four days, followed by xenotransplantation into ∼30 zebrafish larvae for an additional three days of in vivo co-growth prior to confocal imaging. B) Bar graph illustrating the percentage of tumor growth relative to day 0 of transplantation, with each data point representing an individual larva. AA clones harbor significantly higher fitness. **P-value < 0.005. paired t-test. C) TT and AA clones were separately xeno-transplanted into zebrafish larvae, which were followed for three days and imaged by confocal microscopy. D) Quantitative analysis of tumor growth indicates a significantly faster growth rate for AA than TT cells in vivo . Each data dot represents one larva. **P-value < 0.005. paired t-test.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Clone Assay, Transduction, Confocal Microscopy, Transplantation Assay, Expressing, Cell Culture, In Vitro, In Vivo, Imaging

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: A-B) Survival probability from TCGA data plotted after stratifying patients based on the rs1053639 genotype and under a recessive model (A = reference allele (T); B = alternative allele (A)). C ) Correlation between DDIT4 protein and mRNA levels in cancer cell lines from the DepMap database. D) Genotyping results for rs1053639 in seven colon cancer samples. The IHC score was obtained by analyzing the slides by two certified pathologists and applying also a custom script in Fiji-see methods-. E) Representative images of IHC DDIT4 staining. -N and -T after the sample code refer to normal colon mucosa or cancer tissue, respectively. The sensitivity of IHC staining was verified by paraffin embedding pellets from our edited HCT116 cell clones (not shown). Images of sample 04747-N and -T, genotype TT, show high expression of cytoplasmic DDIT4 (E1 and E2). Sample 14809-N, genotype AA, shows a low expression of cytoplasmic DDIT4 (E3), while the 14809-T tumor counterpart shows high expression (E4). Finally, images of sample 07061-N and -T, genotype AT, both show low cytoplasmic expression of the DDIT4 protein (E5 and E6).

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: Staining, Immunohistochemistry, Clone Assay, Expressing

Journal: bioRxiv

Article Title: A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

doi: 10.1101/2025.04.02.646512

Figure Lengend Snippet: Scheme of the approach taken to identify tranSNPs and of the results obtained with rs1053639 in DDIT4 with the HCT116 cell model.

Article Snippet: Antigens were unmasked with Bond Epitope Retrieval Solution 1 Leica AR9961 and incubated with the following antibody (diluted with Bond Primary Antibody Diluent, AR9352 Leica): DDIT4/REDD1 at dilution 1:400 (Proteintech 10638-1-AP).

Techniques: