rragd antibody  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: LINC00622 transcriptionally promotes RRAGD to repress mTORC1-modulated autophagic cell death by associating with BTF3 in cutaneous melanoma

doi: 10.1038/s41419-025-07828-1

Figure Lengend Snippet: A LINC00622 and RRAGD RNA expression was detected by qPCR after knockdown of LINC00622 by siRNAs in melanoma cells. B Western blot detection of the levels of RRAGD, p-mTOR, p-S6K, LCB-II and P62 after LINC00622 depletion. C qPCR detection of LINC00622 expression after LINC00622 overexpression. D Western blot detection of the levels of RRAGD, p-mTOR, p-S6K, LCB-II and P62 after LINC00622 overexpression. The role of LINC00622 in regulating autophagy was validated using autophagy inhibitors 3-MA and MYH1485 treatments in melanoma cells following LINC00622 silencing by Western blot detection of LC3B and P62 ( E , F ) and IF staining of LC3B ( G ). The role of LINC00622 in regulating autophagy was validated using autophagy activator Rapamycin following LINC00622 overexpression by Western blot detection of LC3B and P62 ( H ) and IF staining of LC3B ( I ). Scale bar, 50 µM.

Article Snippet: The following antibodies were used: RRAGD (Cell Signaling Technology, 4470,1:100), LC3B (PTMBIO, PTM-6384 1:100), P62/SQSTM1 (PTMBIO, PTM-6234,1:100).

Techniques: RNA Expression, Knockdown, Western Blot, Expressing, Over Expression, Staining

Journal: Cell Death & Disease

Article Title: LINC00622 transcriptionally promotes RRAGD to repress mTORC1-modulated autophagic cell death by associating with BTF3 in cutaneous melanoma

doi: 10.1038/s41419-025-07828-1

Figure Lengend Snippet: A RRAGD RNA expression was detected by qPCR after knockdown of RRAGD by RNA interference in SK-MEL-28 melanoma cells. Measurement of cell proliferation by CCK-8 assay ( B ), colony formation assay ( C ), Transwell migration assay ( D ) were performed. E RRAGD, p-mTOR, p-S6K, LC3B and P62 were detected by Western blot in SK-MEL-28 melanoma cells after RRAGD depletion. F Sytox green staining was performed to detect the major type of cell death induced by LINC00622 depletion in melanoma cells together with 3-MA, MYH1485, Z-VAD-FMK or Nec-1 treatments. Scale bar, 100 μM. G Evaluate the role of LINC00622 in repressing Rapamycin-induced cell death in melanoma cells by Sytox green staining. Scale bar, 100 μM. H Transmission electron microscopy (TEM) was performed to detect the type of cell death in melanoma cells. Blue arrows indicate autophagosomes. Typical features of autophagic cell death includes the formation of empty vacuoles (EVs) indicated by black arrows and perinuclear space (PNS) showing the separation of outer nuclear membrane (ONM) and inner nuclear membrane (INM) indicated by red arrows. Scale bar, 2 μM.

Article Snippet: The following antibodies were used: RRAGD (Cell Signaling Technology, 4470,1:100), LC3B (PTMBIO, PTM-6384 1:100), P62/SQSTM1 (PTMBIO, PTM-6234,1:100).

Techniques: RNA Expression, Knockdown, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Western Blot, Staining, Transmission Assay, Electron Microscopy, Membrane

Journal: Cell Death & Disease

Article Title: LINC00622 transcriptionally promotes RRAGD to repress mTORC1-modulated autophagic cell death by associating with BTF3 in cutaneous melanoma

doi: 10.1038/s41419-025-07828-1

Figure Lengend Snippet: A The chromatographic analysis of the peptide mixes pulled down by LINC00622. Arrow indicates the identified BTF3 peptide peak in the LINC00622-pulldown sample, which is lack in control EGFP sample. B Biotin-labeled LINC00622 transcript was used to retrieve interacting protein partners by RNA pulldown with beads only and EGFP RNA as controls in protein mix from melanoma cells. Western blot detection showed LINC00622 specific associates with BTF3 but not SOX13. C RNA immunoprecipitation (RIP) assay was performed using antibodies against BTF3 and SOX13 while IgG was used as control. The retrieved LINC00622 RNA was detected by qPCR. U1 transcript was used as negative control. D Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to examine the co-localization of LINC00622 (red) and BTF3 (green) in melanoma cells. Scale bars, 20 μm. E Predicted binding site of BTF3 (diamond) at the promoter region of RRAGD by rVista ( https://rvista.dcode.org/ ). F The binding enrichment of BTF3 at the predicted binding site on RRAGD locus was detected by ChIP-qPCR after knockdown of BTF3 or LINC00622.

Article Snippet: The following antibodies were used: RRAGD (Cell Signaling Technology, 4470,1:100), LC3B (PTMBIO, PTM-6384 1:100), P62/SQSTM1 (PTMBIO, PTM-6234,1:100).

Techniques: Control, Labeling, Western Blot, RNA Immunoprecipitation, Negative Control, Fluorescence, In Situ Hybridization, Immunofluorescence, Binding Assay, ChIP-qPCR, Knockdown

Journal: Cell Death & Disease

Article Title: LINC00622 transcriptionally promotes RRAGD to repress mTORC1-modulated autophagic cell death by associating with BTF3 in cutaneous melanoma

doi: 10.1038/s41419-025-07828-1

Figure Lengend Snippet: A BTF3 RNA expression was detected by qPCR after knockdown of BTF3 by RNA interference in SK-MEL-28 melanoma cells. Measurement of cell proliferation by CCK-8 assay ( B ), colony formation assay ( C ), Transwell migration assay ( D ) were performed. ( E ) BTF3 and RRAGD were detected by qPCR after knockdown of BTF3 by RNA interference. F BTF3, RRAGD, p-mTOR, p-S6K, LC3B and P62 were detected by Western blot in SK-MEL-28 melanoma cells after LINC00622 depletion. Proliferative capacity ( G ) and colony formation ( H ) assays in melanoma cells in response to silencing of LINC00622 could be rescued by overexpression of BTF3. I The enhanced LC3-II generation together with decreased P62 and RRAGD levels after LINC00622 depletion could be significantly tuned back by BTF3 overexpression.

Article Snippet: The following antibodies were used: RRAGD (Cell Signaling Technology, 4470,1:100), LC3B (PTMBIO, PTM-6384 1:100), P62/SQSTM1 (PTMBIO, PTM-6234,1:100).

Techniques: RNA Expression, Knockdown, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Western Blot, Over Expression

Journal: Cell Death & Disease

Article Title: LINC00622 transcriptionally promotes RRAGD to repress mTORC1-modulated autophagic cell death by associating with BTF3 in cutaneous melanoma

doi: 10.1038/s41419-025-07828-1

Figure Lengend Snippet: A Loss of LINC00622 inhibited melanoma growth in a mouse xenograft model. Tumor volumes (mm 3 ) were plotted according to day. B , C The mice were sacrificed at the end of the experiment and the dissected xenografts were shown. Black and white arrows respectively indicate the siNC-treated and siLINC00622-treated xenografts. D The weight comparison of xenografts between siNC-treated and siLINC00622-treated groups. E The expression of LINC00622, RRGAD was detected in dissected xenografts by qPCR. Statistical data of qPCR represented the average of four independent experiments ± SE. F The expression levels of LC3B, P62, and RRAGD in tumor sections were evaluated using IHC staining. Scale bar, 50 µm. G The protein levels of p-mTOR, p-S6K, LC3B, P62, and RRAGD were detected in xenografts after LINC00622 knockdown by Western blot. H A model depicts that LINC00622 transcriptionally enhances RRAGD expression to repress autophagic cell death by associating with BTF3 to promote cutaneous melanoma progression.

Article Snippet: The following antibodies were used: RRAGD (Cell Signaling Technology, 4470,1:100), LC3B (PTMBIO, PTM-6384 1:100), P62/SQSTM1 (PTMBIO, PTM-6234,1:100).

Techniques: Comparison, Expressing, Immunohistochemistry, Knockdown, Western Blot

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 5 IL4 induces RRAGD expression and combined IL4 treatment and BCR crosslinking hyperactivates mTOR signaling. A–C Lymphoma cell line pools transduced with lentiviral CRISPR-Cas9 guide pools targeting AAVS (control) or RRAGD, were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. D Non-malignant normal human LN derived B cells were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. E, F Non-malignant normal human LN derived B cells (N = 4) were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were left untreated or treated ex vivo with IL4, anti-IgM or both as indicated. Cell lysates were prepared for immunoblotting using the indicated epitopes. G densitometry results for p-p70- S6K and RRAGD normalized to actin for all conditions for four primary human LN-derived B lymphocyte samples (# 111, 89, 94, and 95) combined. Left panel: One-way Anova with post hoc Tukey’s test. Right panel: paired nonparametric Wilcoxon test. *p < 0.05, ***p < 0.001. IL4 interleukin 4, BCR B cell receptor, AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Expressing, Transduction, CRISPR, Control, Ex Vivo, Western Blot, Derivative Assay, Virus

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 6 RRAGD is required for S6 Kinase phosphorylation in lymphoma. A The RRAGD gene or AAVS (control) was targeted with lentivirus carrying pooled CRISPR-Cas9 guides in four lymphoma cell lines and following puromycin selection the pools were analyzed for RRAGD protein expression by immunoblotting. B, C RRAGD −/−or AAVS targeted lymphoma cell line pools were treated with anti-IgM or anti-IgG for 10’, cell lysates were made, and protein prepared for immunoblotting using the indicated epitopes. Densitometry data for mean p-p70- S6K:HSP90 for AAVS control cells are shown, while signals for RRAGD −/−cells were close to background. One-Way ANOVA with post hoc Tukey’s test. Densitometry based on three cell lines and N = 2 repeats *p < 0.05, **p < 0.01. D–F RRAGD −/−or AAVS targeted lymphoma cell line pools were treated +/−IL4 for 6 h and +/−anti-IgM for 10’, cell lysates were made and protein prepared for immunoblotting using the indicated epitopes. D A representative blot for OCI-LY7. E Densitometry of mean p-4E-BP1:total 4E-BP1 for AAVS control cells +/−IL4 for 6 h and +/−anti-IgM/G. One-Way ANOVA with post hoc Dunn’s test. F Densitometry of comparative data for AAVS and RRAGD −/−cells; Mann–Whitney test. Densitometry based on three cell lines and N = 2 repeats. ns, not significant, *p < 0.05, **p < 0.01. AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, IgG immunoglobulin G, IL4 interleukin 4, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Phospho-proteomics, Control, CRISPR, Selection, Expressing, Western Blot, MANN-WHITNEY, Virus

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 7 STAT6 mutations hyperinduce RRAGD expression and IL4 and BCR induced mTOR signaling. Three lymphoma cell lines were lentivirally transduced with cDNA/ORFs for WT or MUT STAT6 and cells sorted via GFP fluorescence. Cells were stimulated with IL4 and BCR crosslinking as indicated or left untreated and detergent lysates prepared for immunoblotting using the indicated epitopes. A–C Representative immunoblotting results for N = 2 independent experiments per cell pool. D Results from densitometry for phospho-S6K normalized to HSP90 for indicated conditions across three cell lines (N = 6). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05. E Results from densitometry for RRAGD normalized to HSP90 for indicated conditions for N = 2 independent experiments across three cell lines (N = 6 measurements). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05; **p < 0.01. STAT6 Signal Transducer and Activator of Transcription 6, IL4 interleukin 4, WT wild type, MUT mutated, GFP green fluorescence protein, cDNA complementary DNA, ORFs open reading frames, IL4 interleukin 4, BCR B cell receptor, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Expressing, Transduction, Western Blot