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RHOQ interacts with PAK1 and promotes its phosphorylation. (A) Immunoprecipitation assay for the binding of HA‐PAK1 to flag‐RHOQ, flag‐RHOQ(Q67L) and flag‐RHOQ(T23N) in Huh7 cells. Immunoprecipitation was performed using anti‐HA <t>and</t> <t>anti‐flag</t> antibodies, and immunoblotting analysis was performed using PAK1 and RHOQ antibodies. (B) Immunoprecipitation assay for the binding of flag‐RHOQ to HA‐PAK1 and HA‐del‐PAK1 in Huh7 cells. (C) Immunoblotting analysis for PAK1 and del‐PAK1 phosphorylation levels in Huh7 cells. (D) Molecular docking simulates the binding of RHOQ to PAK1; green represents RHOQ, and blue represents PAK1. Yellow dashed lines represent hydrogen bonds. HDOCK web server: http://hdock.phys.hust.edu.cn/ . (E) Immunoblotting analysis for the PAK1 downstream effectors and their phosphorylation in Huh7 cells after IPA‐3 (10 µM) treatment. (F) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. IPA‐3 concentration was 10 µM. (G) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. FTY720 concentration was 10 µM. (H) Immunoblotting analysis for ATF6 downstream effectors and their phosphorylation in Huh7 cells. (I) Significantly enriched pathways ( Q < .05) are shown. The x‐axis indicates the Gene Ratio, while the y ‐axis shows the pathway categories. Bubble size reflects the gene count in each pathway, and bubble colour denotes the padj (FDR‐adjusted p value). (J) Represented images for transwell assay of Huh7 cells. IPA‐3 concentration was 10 µM. (K) Represented images for transwell assay of Huh7 cells. FTY720 concentration was 10 µM.
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RHOQ interacts with PAK1 and promotes its phosphorylation. (A) Immunoprecipitation assay for the binding of HA‐PAK1 to flag‐RHOQ, flag‐RHOQ(Q67L) and flag‐RHOQ(T23N) in Huh7 cells. Immunoprecipitation was performed using anti‐HA and anti‐flag antibodies, and immunoblotting analysis was performed using PAK1 and RHOQ antibodies. (B) Immunoprecipitation assay for the binding of flag‐RHOQ to HA‐PAK1 and HA‐del‐PAK1 in Huh7 cells. (C) Immunoblotting analysis for PAK1 and del‐PAK1 phosphorylation levels in Huh7 cells. (D) Molecular docking simulates the binding of RHOQ to PAK1; green represents RHOQ, and blue represents PAK1. Yellow dashed lines represent hydrogen bonds. HDOCK web server: http://hdock.phys.hust.edu.cn/ . (E) Immunoblotting analysis for the PAK1 downstream effectors and their phosphorylation in Huh7 cells after IPA‐3 (10 µM) treatment. (F) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. IPA‐3 concentration was 10 µM. (G) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. FTY720 concentration was 10 µM. (H) Immunoblotting analysis for ATF6 downstream effectors and their phosphorylation in Huh7 cells. (I) Significantly enriched pathways ( Q < .05) are shown. The x‐axis indicates the Gene Ratio, while the y ‐axis shows the pathway categories. Bubble size reflects the gene count in each pathway, and bubble colour denotes the padj (FDR‐adjusted p value). (J) Represented images for transwell assay of Huh7 cells. IPA‐3 concentration was 10 µM. (K) Represented images for transwell assay of Huh7 cells. FTY720 concentration was 10 µM.

Journal: Clinical and Translational Medicine

Article Title: ATF6‐mediated GTPase RHOQ promotes liver cancer progression via enhancing aerobic glycolysis through PAK1 phosphorylation

doi: 10.1002/ctm2.70871

Figure Lengend Snippet: RHOQ interacts with PAK1 and promotes its phosphorylation. (A) Immunoprecipitation assay for the binding of HA‐PAK1 to flag‐RHOQ, flag‐RHOQ(Q67L) and flag‐RHOQ(T23N) in Huh7 cells. Immunoprecipitation was performed using anti‐HA and anti‐flag antibodies, and immunoblotting analysis was performed using PAK1 and RHOQ antibodies. (B) Immunoprecipitation assay for the binding of flag‐RHOQ to HA‐PAK1 and HA‐del‐PAK1 in Huh7 cells. (C) Immunoblotting analysis for PAK1 and del‐PAK1 phosphorylation levels in Huh7 cells. (D) Molecular docking simulates the binding of RHOQ to PAK1; green represents RHOQ, and blue represents PAK1. Yellow dashed lines represent hydrogen bonds. HDOCK web server: http://hdock.phys.hust.edu.cn/ . (E) Immunoblotting analysis for the PAK1 downstream effectors and their phosphorylation in Huh7 cells after IPA‐3 (10 µM) treatment. (F) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. IPA‐3 concentration was 10 µM. (G) Immunoblotting analysis for RHOQ downstream effectors and their phosphorylation in Huh7 cells. FTY720 concentration was 10 µM. (H) Immunoblotting analysis for ATF6 downstream effectors and their phosphorylation in Huh7 cells. (I) Significantly enriched pathways ( Q < .05) are shown. The x‐axis indicates the Gene Ratio, while the y ‐axis shows the pathway categories. Bubble size reflects the gene count in each pathway, and bubble colour denotes the padj (FDR‐adjusted p value). (J) Represented images for transwell assay of Huh7 cells. IPA‐3 concentration was 10 µM. (K) Represented images for transwell assay of Huh7 cells. FTY720 concentration was 10 µM.

Article Snippet: The remaining portion was incubated overnight at 4°C with anti‐flag tag antibody (Proteintech, China), anti‐HA tag antibody (Proteintech) and isotype IgG (Beyotime).

Techniques: Phospho-proteomics, Immunoprecipitation, Binding Assay, Western Blot, Analysis, Docking Assay, Concentration Assay, Transwell Assay