IL4R mutations in PMBCL and their ability to induce constitutive phosphorylation of STAT6 in HEK293 cells. (A) Distribution of IL4R mutations in PMBCL cell lines (n = 3) (○ and ⃞ ), previously reported in Gunawardana et al14 and PMBCL clinical samples (n = 62) (●) identified by targeted deep amplicon and Sanger sequencing. Missense and frameshift mutations are represented by circles and squares, respectively. Variations in noncoding regions, reported single nucleotide polymorphisms, and synonymous mutations are not shown. (B-C) HEK293-STAT6 cells were transfected with IL4R WT, IL4R mutants, or mock expression vector (empty) and either left UT or stimulated with human recombinant IL-4 (0.1 ng/mL) for 24 hours. (B) Western blot analysis of IL4R, pSTAT6, STAT6, and GAPDH in cell lysates. (C) STAT6-dependent SEAP released in cell-free supernatants. The values were normalized to untreated HEK293-STAT6 cells transfected with IL4R WT vector. (C) Mean ± SD of 3 independent experiments; significance was evaluated using 1-way ANOVA followed by Bonferroni post test. *P < .05; ***P < .001, ****P < .0001. SD, standard deviation; UT, untreated; UTR, untranslated region.
