Fig 1: Isobutyric acid (IBA) physically binds to RACK1. (A) Mass spectrometry analysis of a Venn diagram. (B) In vitro pulldown assay analyzing the interaction between IBA and RACK1. (C) Immunofluorescence analysis detected colocalization of IBA (in red) with RACK1 protein (in green) in HCT116 and SW48 cells; scale bar = 20 μm. (D) Cellular thermal shift assay analyzed the effect of IBA on RACK1 stability. (E) Predicted amino acid sites where IBA may bind with RACK1.
Fig 2: Isobutyric acid (IBA) promotes colorectal cancer (CRC) cell metastasis mediated by the RAKC1 signaling pathway. (A) The effect of IBA on the migration of HCT116 CRC cells after knocking down RACK1 analyzed by scratch assay. (B) The effect of IBA on the migration of SW48 CRC cells after knocking down RACK1 analyzed by scratch assay. (C) The effect of knocking down RACK1 on the migration and invasion of HCT116 CRC cells after treatment with IBA analyzed by Transwell migration and Matrigel invasion assay. (D) The effect of knocking down RACK1 on the migration and invasion of SW48 CRC cells after treatment with IBA analyzed by Transwell migration and Matrigel invasion assay. Scale bar = 100 μm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 3: Inhibition of RACK1 promotes tumor metastasis by eliminating isobutyric acid (IBA) in vivo. (A) Mouse liver metastasis tissue of HCT116. (B) Statistics of mouse liver metastatic tumors of HCT116. (C) H&E images of mouse liver metastasis of HCT116. (D) Mouse lung metastasis tissue of HCT116. (E) Statistics of mouse lung metastatic tumors of HCT116. (F) H&E images of mouse lung metastasis of HCT116. (G) Immunohistochemistry (IHC) staining results of mouse liver metastasis of HCT116. Scale bar = 50 μm; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 4: Isobutyric acid (IBA) affects RAKC1 signaling pathway activation. (A, B) Protein blot analysis of the effect of IBA on RACK1 expression in HCT116 (A) and SW48 (B) cells. (C, D) RT‐qPCR analysis of the effect of IBA on RACK1 mRNA expression in HCT116 (C) and SW48 (D) cells. (E, F) Protein blot analysis of the effect of IBA on the RACK1 signaling pathway in HCT116 (E) and SW48 (F) cells. (G–J) RT‐qPCR analysis of the effect of IBA on RACK1 signaling pathway mRNA expression in HCT116 (G, H) and SW48 (I, J) cells. (K–N) RT‐qPCR (K, L) and protein blot (M, N) analysis confirming RACK1 gene knockdown in HCT116 and SW48 cells. (O) Protein blot analysis demonstrating that IBA affects the RACK1 signaling pathway through RACK1 in HCT116 cells. (P) Protein blot analysis demonstrating that IBA affects the RACK1 signaling pathway through RACK1 in SW48 cells. NS, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 5: Schematic diagram of isobutyric acid (IBA) promoting colorectal cancer (CRC) metastasis through RACK1 activation. IBA directly interacts with RACK1 and activates downstream AKT and FAK phosphorylation, thereby increasing the transcriptional activity of MMP2, MMP9, and ICAM1, promoting CRC metastasis.
Supplier Page from ABclonal Technology for RACK1 Rabbit mAb