wheat germ agglutinin conjugated to fitc fitc wga  (Santa Cruz Biotechnology)

Journal: Autophagy

Article Title: Dual mechanism of human ATG10S in suppressing MEF2A-Driven pro-inflammatory responses

doi: 10.1080/15548627.2026.2676074

Figure Lengend Snippet: ATG10S promotes complete autophagic flux and inhibits pro-inflammatory cytokine genes transcription. (A, B) in S protein- or LPS-induced pro-inflammatory cell models, autophagic flux in co-cultured MDMs and BEAS-2B cells was evaluated using the tandem fluorescent sensor RFP-GFP-LC3B. Control groups were not treated with S protein, LPS, or ATG10S. A representative image from one of eight randomly selected fields (20 cells per field) is shown for each group. Scale bar: 15 μm. (C, D) in S protein- or LPS-induced pro-inflammatory zebrafish and cell models, lysates from larvae and from co-cultured MDMs and BEAS-2B cells were immunoprecipitated with an anti-LC3B antibody to assess interactions between LC3B and LAMP2, ATG5–ATG12, ATG7, or SQSTM1. A representative blot from five independent experiments is shown. (E, F) in S protein- or LPS-induced pro-inflammatory cell models, luciferase assays were performed to evaluate the effect of ATG10S on the transcriptional activity of pro-inflammatory cytokine gene promoters ( IFNA , IFNG , IL1B , IL6 , TNF , IL8 , and CCL2 ) in co-cultured MDMs. *** p < 0.001. Data were presented as mean ± sd ( n = 3) (e). DNA IP assays were performed to assess the binding of ATG10S to pro-inflammatory cytokine gene promoters. lysates from co-cultured MDMs were immunoprecipitated using an anti-His antibody (specific to His-tagged ATG10S), and promoter enrichment was quantified by qRT-PCR. Data are presented as mean ± sd ( n = 3) (f).

Article Snippet: Antibody against LAMP2 (sc-18822) was obtained from Santa Cruz Biotechnology.

Techniques: Cell Culture, Control, Immunoprecipitation, Luciferase, Activity Assay, Binding Assay, Quantitative RT-PCR

Journal: Autophagy

Article Title: Dual mechanism of human ATG10S in suppressing MEF2A-Driven pro-inflammatory responses

doi: 10.1080/15548627.2026.2676074

Figure Lengend Snippet: ATG10S promotes autolysosome formation to facilitate MEF2A degradation. (A) in S protein- or LPS-induced pro-inflammatory zebrafish and cell models, MDMs and zebrafish lysates were subjected to co-IP with an anti-LC3B antibody to examine the effect of ATG10S on the interaction between LC3B, LAMP2, and MEF2A. Representative blots from five independent experiments are shown. (B) in S protein- or LPS-induced pro-inflammatory cell models, immunofluorescence was performed to assess the subcellular colocalization of MEF2A and LC3B after ATG10S treatment in MDMs. Control groups were untreated with S protein, LPS, or ATG10S. A representative image from one of eight observed fields (20 cells per field) is shown for each group. Scale bar: 15 μm. (C, D) in S protein- or LPS-induced pro-inflammatory cell models, co-IP (C) using an anti-HA antibody was performed to assess the interaction between LC3B and either wild-type MEF2A or the MEF2A∆LIR mutant in MDMs. A representative blot from five independent experiments is shown. Immunofluorescence (d) with anti-HA and anti-LC3B antibodies was used to visualize subcellular colocalization between LC3B and either MEF2A or MEF2A∆LIR in MDMs. A representative image from one of eight observed fields (20 cells per field) is shown for each group. Scale bar: 15 μm. (E, F) in S protein- or LPS-induced pro-inflammatory cell models, western blotting was used to examine MEF2A protein levels in MDMs treated with 2.5 μM ATG10S, 10 μM CQ, or 10 μM MG132. A representative blot from five independent experiments is shown, and statistical analysis was performed. *** p < 0.001. Data are presented as mean ± sd ( n = 5) (e). Luciferase assays were performed to assess the effect of ATG10S on the transcriptional activity of pro-inflammatory cytokine gene promoters in MDMs treated with 10 μM CQ, which inhibits autolysosomal degradation. Data are presented as mean ± sd ( n = 3) (f).

Article Snippet: Antibody against LAMP2 (sc-18822) was obtained from Santa Cruz Biotechnology.

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Control, Mutagenesis, Western Blot, Analysis, Luciferase, Activity Assay

Journal: Autophagy

Article Title: Dual mechanism of human ATG10S in suppressing MEF2A-Driven pro-inflammatory responses

doi: 10.1080/15548627.2026.2676074

Figure Lengend Snippet: The MEF2 domain of MEF2A mediates its direct interaction with ATG10S. (A) in S protein- or LPS-induced pro-inflammatory cell models, endogenous ATG10S was knocked down in MDMs. Co-IP with an anti-LC3B antibody was performed to assess the effect of ATG10S on the interaction between LC3B, LAMP2, and MEF2A. A representative blot from five independent experiments is shown. (B) in S protein- or LPS-induced pro-inflammatory zebrafish and cell models, co-IP was performed using an anti-His antibody to detect interactions between ATG10S (with His tag) and MEF2A, LAMP2, and LC3B in MDMs and zebrafish. A representative blot from five independent experiments is shown. (C, D) in S protein- or LPS-induced pro-inflammatory cell models, subcellular colocalization of MEF2A and ATG10S in MDMs was assessed using immunofluorescence. Cells were stained with anti-MEF2A (green) and anti-His (red) antibodies. A representative image from eight observed fields (20 cells per field) is shown for each group. Scale bars: 15 μm (C). MDMs were subjected to nucleus/cytoplasm fractionation followed by western blotting to analyze MEF2A distribution. Co-IP with an anti-His antibody was conducted to detect the interaction between ATG10S and MEF2A in nuclear and cytoplasmic fractions. A representative blot from five independent experiments is shown. HSP90 and LMNB1 were used as cytoplasmic and nuclear markers, respectively (d). (E) a representative SPR sensorgram showing the binding of ATG10S (at 133, 266, 531, 1062, and 2125 nM) to MEF2A immobilized on the chip (27 μg/mL), confirming a concentration-dependent direct interaction. (F) Schematic representation of the functional domains of full-length MEF2A and its deletion mutant (∆MEF2A). (G) in S protein-induced pro-inflammatory cell models, co-IP with an anti-His antibody was conducted to evaluate the interaction between ATG10S and either full-length MEF2A or ∆MEF2A in MDMs. (H, I) in S protein- or LPS-induced pro-inflammatory cell models, DNA IP assays were performed to assess the binding of HA-MEF2A or HA-∆MEF2A to the promoters of IFNA , IFNG , IL1B , IL6 , TNF , IL8 , and CCL2 genes, and to evaluate the effect of ATG10S on this binding. MDMs lysates were immunoprecipitated with an anti-HA antibody, and promoter sequences were detected by qRT-PCR (H). Luciferase assays were performed to evaluate the transcriptional activity of MEF2A or ∆MEF2A on pro-inflammatory gene promoters, and to determine the effect of ATG10S on this activity. *** p < 0.001, ### p < 0.001. Data are presented as mean ± sd ( n = 3) (i).

Article Snippet: Antibody against LAMP2 (sc-18822) was obtained from Santa Cruz Biotechnology.

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Fractionation, Western Blot, Sensorgram, Binding Assay, Concentration Assay, Functional Assay, Mutagenesis, Immunoprecipitation, Quantitative RT-PCR, Luciferase, Activity Assay