Fig. 5: The identified humoral antigens in the focal adhesion complexes are ribonucleoproteins (RNPs) colocalized with ribosomal translation machinery.

a GO enrichment analysis of the five protein antigens correlated with focal adhesion complexes. b Fluorescent staining of single-strand nucleotides using acridine orange in MKN1 cells. The blue color represents Hoechst nuclear staining. DIC, differential interference contrast image. c Fluorescent dual staining of anti-FAK antibody (red) and Cy5-conjugated oligo-dT probe (green) in human gastric cancer cells MKN1 and GSU, a lung adenocarcinoma cell A549, and a pancreatic cancer cell PANC-1. The blue color represents Hoechst staining. The white bars indicate 20 μm. d Fluorescent immunocytochemistry of FAK (red) and a ribosome marker (ribosomal protein S6 [RPS6]) (green). The blue represents nuclear staining. The white bars indicate 20 μm. b–d White arrowheads indicate representative colocalization signals, and the similar colocalizations were reproducibly observed in all analyzed cells with formations of visible focal adhesions. e The validity of the anti-FAK antibody in our RIP procedure was confirmed by immunoblots. The arrowhead and asterisk represent the FAK protein and heavy chain of the anti-FAK rabbit antibody, respectively. f The left and right panels show electropherograms of the precipitated RNA and constructed cDNA sequencing library, respectively, of FAK-RIP experiments in biological duplicates (#1 and #2). g Plots of the results of the next-generation sequencing of FAK-RIP. The x and y-axes represent the numbers of sequencing reads of input cellular RNA and the minimum of those in the two FAK-RIP experiments, respectively. Each dot represents each transcript/gene, while red dots indicate the enriched transcripts/genes used in the motif enrichment analysis. h The top five enriched motifs in the transcript sequences among FAK-RIP-enriched genes (354 genes) compared to FAK-RIP-diminished genes (454 genes). i Histograms of the numbers of genes plotted according to the numbers of “CAGCCC” motifs within the gene transcript sequences. The left and right panels represent FAK-RIP-enriched and -diminished gene sets, respectively.