anti flag magnetic beads  (MedChemExpress)

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