anti flag magnetic beads  (MedChemExpress)

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A induces mono-methylation of lysine 193 on IRF3 (A) HEK293T cells were transfected with FLAG-IRF3 and/or HA-KMT5A plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads. Subsequent analysis was conducted via IB. (B) Whole-cell lysates were extracted from RKO cells with either control (shNC) or KMT5A shRNA (#1 and #2) silencing. IP was performed using anti-IRF3 antibodies, followed by WB analysis. (C) RKO cells were transfected with HA-KMT5A WT or HA-KMT5A D338A, IP was performed using anti-IRF3 antibodies, followed by WB analysis. (D) RKO cells were treated with either DMSO or UNC0379. IP was performed using anti-IRF3 antibodies, followed by IB analysis. (E) RKO cells were treated with either DMSO or varying concentrations of UNC0379. IP was conducted using anti-IRF3 antibodies, followed by IB analysis. (F) The levels of IFN-β in RKO cells from experiment (E) were quantified using ELISA. Data were analyzed using one-way ANOVA with Tukey’s post-hoc test, presented as mean ± SD. Statistical significance was defined as ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. (G) In vitro methylation assays were conducted by incubating purified His-IRF3 with KMT5A in the presence of S-adenosyl-L-methionine, followed by IB analysis. (H) Secondary mass spectrometry results of IRF3 K193 methylation were obtained. (I) HEK293T cells were transfected with FLAG-IRF3 wild-type or mutant plasmids, followed by transfection with either a vector or HA-KMT5A. Whole-cell lysates were collected, and IP was performed using anti-FLAG magnetic beads, followed by IB analysis. (J) Amino acid sequences at the K193 site of IRF3 were compared across different species. All immunoblotting experiments were conducted independently in triplicate, yielding consistent results.

Article Snippet: Anti-Flag magnetic beads , MedChemExpress , CAT#HY-K0207.

Techniques: Methylation, Transfection, Magnetic Beads, Control, shRNA, Enzyme-linked Immunosorbent Assay, In Vitro, Purification, Mass Spectrometry, Mutagenesis, Plasmid Preparation, Western Blot

Journal: iScience

Article Title: KMT5A-mediated methylation of IRF3 promotes tumor progression through immune suppression

doi: 10.1016/j.isci.2026.116902

Figure Lengend Snippet: KMT5A hindering production of IFN-β depends on IRF3 K193 methylation (A) HEK293T cells were transfected with vector, FLAG-IRF3 wild-type, or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent analysis was performed via IB. (B) KMT5A-knockout RKO cells were transfected with FLAG-IRF3 wild-type or K193R mutant plasmids. The cells were treated with poly(I) or UNC0379, then whole-cell lysates were collected, and IP was conducted using anti-FLAG magnetic beads, followed by IB analysis. (C) RKO cells from experiment (B) were co-transfected with reporter plasmids IFN-β-Luc and pRL-TK. After 24 h, the cells were harvested, and luciferase activity was measured using a dual-luciferase reporter assay kit. (D) The relative mRNA levels of INF-β in RKO cells from experiment (B) were quantified using qPCR. (E) KMT5A knockout RKO cells were transfected with HA-KMT5A and FLAG-IRF3 wild-type or K193R mutant plasmids. Whole-cell lysates were collected, followed by IP using anti-FLAG magnetic beads, and subsequent IB analysis was performed. (F) Relative quantification of phosphorylated IRF3 (p-IRF3) protein was conducted in experiment (E). (G) The relative mRNA levels of INF-β in RKO cells from experiment F were quantified using qPCR. (C) and (D) were analyzed using one-way ANOVA with Tukey’s post-hoc test, with data presented as mean ± SD. (F) and (G) were analyzed using two-way ANOVA with Tukey’s post-hoc test. Statistical significance was defined as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, while ns indicates no statistical difference. All immunoblotting experiments were performed independently in triplicate, yielding consistent results.

Article Snippet: Anti-Flag magnetic beads , MedChemExpress , CAT#HY-K0207.

Techniques: Methylation, Transfection, Plasmid Preparation, Mutagenesis, Magnetic Beads, Knock-Out, Luciferase, Activity Assay, Reporter Assay, Quantitative Proteomics, Western Blot

Journal: Cancer Communications

Article Title: NSUN2 Promotes Cancer Immune Evasion via Its Moonlighting Function Acting on Metabolic Reprogramming

doi: 10.34133/cancomm.0042

Figure Lengend Snippet: NSUN2 acted as a cofactor of GATA3 and promoted the expression of SUCLG1/SUCLG2 and chemokines. (A) Schematic representation of the critical regulatory proteins associated with succinate metabolism. (B) Heatmap showing the differential expression of succinate-related metabolic genes in sorted mCherry + CD45 − tumor cells isolated from subcutaneous tumors of the EV and Nsun2 MUT groups (data from Fig. A). (C) RT-qPCR analysis of the expression levels of the succinate-related genes in sg Ctrl , sg Nsun2 , and sg Nsun2 + Nsun2 MUT -reconstituted MC38 cells; n = 3 samples for each group. (D) Schematic representation of anti-Flag antibody-mediated IP and MS analysis of Flag- NSUN2 MUT and EV group samples. (E) Heatmap depicting differentially abundant proteins identified through MS analysis in HEK293T cells expressing Flag- NSUN2 MUT compared with control EV groups ( n = 2 per group). (F) WB analysis of whole-cell lysate and IP from HEK293T cells transfected with Flag- NSUN2 MUT , HA-GATA3, or both in combination. IP was performed with anti-HA or anti-Flag antibodies, followed by WB with the indicated antibodies to detect the physical interaction between NSUN2 MUT and GATA3. (G) Representative IF staining images of NSUN2 and GATA3 in MC38 cells. (H) Predicted GATA3-binding sites on SUCLG1 and SUCLG2 promoters. (I) ChIP–qPCR analysis of GATA3 binding to the promoters of SUCLG1 , SUCLG2 , CCL2 , CCL5 , and CCL7 promoters in HEK293T cells; n = 3 samples for each group. (J) Schematic representation of truncated forms of NSUN2 and GATA3. WB analysis of WCL and anti-Flag or anti-HA IP from HEK293T cells transfected with the MTD, TA, and ZNF constructs. (K) Luciferase reporter analysis of the transcriptional activation of SUCLG1 and SUCLG2 mediated by NSUN2 , its ΔMTD variants, NSUN2 MUT , or the corresponding ΔMTD variants. (L) Indirect IF of p65, p50, and DAPI in MC38 cells treated with succinate or DMSO. Results are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant through an unpaired t test. Abbreviations: ChIP–qPCR, chromatin immunoprecipitation–quantitative polymerase chain reaction; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EV, empty vector; GATA3, GATA-binding protein 3; IF, immunofluorescence; IP, immunoprecipitation; MS, mass spectrometry; MTD, methyltransferase domain; MUT, mutant; NSUN2, NOP2/Sun RNA methyltransferase 2; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SD, standard deviation; sg Ctrl , single-guide RNA control; SUCLG1, succinate-CoA ligase GDP/ADP-forming subunit α; SUCLG2, succinate-CoA ligase GDP/ADP-forming subunit β; TA, transactivation domain; WB, western blotting; ZNF, zinc finger domain.

Article Snippet: For IP, cell lysates were incubated overnight at 4 °C with anti-Flag (HY-K0207, MedChemExpress) or anti-HA (HY-K0201, MedChemExpress) magnetic beads, followed by 3 washes with tris-buffered saline with Tween-20 (TBST) buffer and resolution via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

Techniques: Expressing, Quantitative Proteomics, Isolation, Quantitative RT-PCR, Control, Transfection, Staining, Binding Assay, ChIP-qPCR, Construct, Luciferase, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation, Immunofluorescence, Immunoprecipitation, Mass Spectrometry, Mutagenesis, Reverse Transcription, Standard Deviation, Western Blot