ptmscan acetyl-lysine motif kit no. 13416  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: C1q + Macrophage–Tumor Cell Interaction Promoted Tumorigenesis via GPR17/PI3K/AKT Pathway Induced DNA Hypermethylation in Nasopharyngeal Carcinoma

doi: 10.1002/advs.202503434

Figure Lengend Snippet: C1q activated PI3K‐AKT signaling through interaction with membrane GPR17. A) C666‐1 cells were treated with 1 µg mL −1 C1q for 4 h. The immunofluorescence assay was performed to assess the localization of C1q. Dil was stained to identify the cell membrane. B) C666‐1 cells were treated with 1 µg mL −1 C1q for 4 h. Membrane protein was extracted, and the C1q‐interacting protein was pulled down by anti‐C1q antibody. The precipitants were performed with 4‐D label‐free quantitative proteomics to identify C1q coupled protein. C) Pathway enrichment was conducted in the C1q‐interacting proteins. D–H) C666‐1 cells were treated with 1 µg mL −1 C1q for 4 h. (D) The immunoprecipitation assay was performed to analyze the interaction between C1q and GPR17. (E) The interaction between C1q and GPR17 was detected by the immunofluorescence assay. (F) Cellular calcium level was evaluated using flow cytometry analysis. (G) The interaction between GPR17 and PI3K was detected by the immunoprecipitation assay. (H) The immunofluorescence assay was performed to assay the interaction between GPR17 and PI3K. I,J) Indicated cells were treated with 1 µg mL −1 C1q for 4 h. In some cases, the expression of GPR17 in C666‐1 cells was knocked down. (I) The flow cytometry analysis was performed to explore cellular calcium levels. (J) Activation of the PI3K/AKT pathway was detected by western blot analysis. K,L) Indicated cells were treated with 1 µg mL −1 C1q for 24 h. In some cases, the expression of GPR17 in C666‐1 cells was knocked down. (K) The expression of DNMTs was evaluated by RT‐qPCR analysis. (L) The expression of DNMTs was determined by western blot analysis. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented. Two‐tailed unpaired Student's t ‐test was used to analyze the difference between the two groups.

Article Snippet: Antibodies involved in this investigation were as follows: C1q (sc‐53544, Santa Cruz Biotechnology), DNMT1 (24206‐1‐AP, Proteintech, USA), DNMT3A (20954‐1‐AP, Proteintech), DNMT3B (26971‐1‐AP, Proteintech), PI3K (67071‐1‐Ig, Proteintech), AKT (10176‐2‐AP, Proteintech), phospho‐AKT (66444‐1‐Ig, Proteintech), GPR17 (13416‐1‐AP, Proteintech), GAPDH (10494‐1‐AP, Proteintech), phospho‐PI3K (AF3242, Affinity, USA), ATP1A1 (sc‐71639, Santa Cruz Biotechnology), HRP‐conjugated Goat Anti‐Mouse IgG(H + L) (SA00001‐1, Proteintech), HRP‐conjugated Goat Anti‐Rabbit IgG(H + L) (SA00001‐2, Proteintech).

Techniques: Membrane, Immunofluorescence, Staining, Quantitative Proteomics, Immunoprecipitation, Flow Cytometry, Expressing, Activation Assay, Western Blot, Quantitative RT-PCR, Two Tailed Test