Fig. 2

JMJD6 Enhances MAPK Signaling in Melanoma Cells through Regulation of Alternative Splicing. a Bioinformatics analysis of the different AS events that were identified in JMJD6-depleted A375 cells using the DAVID Functional Annotation Tool (DAVID, https://david.ncifcrf.gov/). b RNA-IP assay in A375 cells was performed with IgG or JMJD6 antibody followed by qRT-PCR with primer pairs for the intron-exon junction in PAK1 pre-mRNA. c The transcripts of full length (PAK1) and exon 15-skipped PAK1 (PAK1Δ15) were analyzed by RT-PCR using RNA extracted from control siRNA, siJMJD6#1 or siJMJD6#2-treated A375 cells. Arrows indicate the location of primers for RT-PCR analyses. Quantitation was done by densitometry and expressed as signals of PAK1/PAK1Δ15 ratios. d Schemes illustrating primer and probe design to detect PAK1 and PAK1Δ15 are shown. A375 cells were transfected with control siRNA/vector, siJMJD6#1 or siJMJD6#2, JMJD6, or JMJD6m, and TaqMan assays were performed to determine the ratio of PAK1 to PAK1Δ15 expression. e, f A375 and 451Lu cells were treated with control siRNA, siJMJD6#1, siJMJD6#2, vector, JMJD6 or JMJD6m, and Western blotting analysis was performed with antibodies as indicated. g A schematic representation of the structure of PAK1 and PAK1Δ15. The PBD (p21-binding domain), AID (auto inhibitory domain), and kinase domain are shown. The PAK1Δ15 is lack of 15th exon (517-533 aa) thus has an incomplete kinase domain. h A375 and 451Lu cells were transfected with vector, FLAG-PAK1 or FLAG-PAK1Δ15 expression plasmids, and Western blotting analysis was performed with antibodies as indicated