Fig. 1: FLNa and FLNb are substrates of ASB2α in mouse Th2 lymphocytes.

a Relative expression of ASB2α transcripts during Th2 differentiation of naive CD4⁺ T lymphocytes of ctrl mice assessed by RT-qPCR. b Expression of ASB2α transcripts in ctrl and ASB2 cKO Th2 lymphocytes assessed by RT-qPCR. c, d Mass spectrometry quantitative analyses of protein abundance differences between cell extracts of ctrl and ASB2 cKO Th2 lymphocytes. The volcano plot (c) illustrates for each protein the statistical significance of the variation as a function of the amplitude of the abundance (log2) difference between the two conditions. Dashed lines indicate cut-off values corresponding respectively to a Student t-test p value of 0.01 and a log2-transformed intensity difference of 0.5. Bar plots (d) show the intensities of ASB2, STAT6, GATA3, c-MAF, FLNa, and FLNb in each condition based on the MS measurements. Full data are presented in Supplementary Data 1. e Expression of FLNa was analyzed by intracellular flow cytometry in ctrl and ASB2 cKO Th2 lymphocytes. f Cell extracts of ctrl and ASB2 cKO Th2 lymphocytes were immunoprecipitated with control (Ctrl IP) or anti-FLNa (FLNa IP) antibodies. Pre-cleared cell lysates (input), unbound, and bound fractions were immunoblotted with antibodies to FLNa. After stripping, the blot was reprobed with antibodies to ubiquitylated proteins. One experiment out of two is shown. g Expression of FLNa was analyzed by intracellular flow cytometry in ctrl Th2 lymphocytes treated at day 5 with the PS-341 proteasome inhibitor at 4 nM during 20 h. h Expression of FLNa and FLNb was analyzed by western blot in naive CD4⁺ T cells, Th1, Th2, Th17, and Treg cells generated from naive CD4⁺ T lymphocytes of ctrl mice. i Expression of FLNa was analyzed by intracellular flow cytometry in Th1, Th2, Th17, and Treg cells generated from naive CD4⁺ T lymphocytes of ctrl mice. j Representative flow cytometry and quantification of FLNa expression in Th1 (CD4⁺CXCR3⁺CCR6⁻), Th2 (CD4⁺CXCR3⁻CCR6⁻CRTH2⁺) and Th17 (CD4⁺CXCR3⁻CCR6⁺) lymphocytes of human PBMCs. k Relative expression of ASB2 transcripts in naive human CD4⁺ T lymphocytes and after 5 days of culture in a Th2 or Th1 polarizing medium assessed by RT-qPCR. l Expression of ASB2α was analyzed by western blot in naive human CD4⁺ T lymphocytes and after 3 and 6 days of culture in a Th2 polarizing medium. m Expression of FLNa was analyzed by intracellular flow cytometry in human Th2 lymphocytes treated at day 5 with the PS-341 proteasome inhibitor at 10 nM during 20 h. n Gene set enrichment analyses of genes significantly up- and down-regulated in ctrl Th2 lymphocytes upon stimulation performed using transcriptomes of stimulated vs non-stimulated ASB2 cKO Th2 lymphocytes. Data are mean ± SEM of biological replicates. Sample size, d1 = 6, d2 = 6, d3 = 14, d4 = 11, d5 = 9 and d6 = 14 for (a); ctrl = 28 and cKO = 32 for (b); ctrl = 9 and cKO = 9 for (c); ctrl = 9 and cKO = 9 for (d); ctrl = 22 and cKO = 21 for (e); ctrl = 8 for (g); Th1 = 9, Th2 = 9, Th17 = 6 and Treg = 6 for (i); n = 8 for (j); naive = 4, Th2 = 4 and Th1 = 3 for (k); n = 4 for (l); and n = 7 for (m). p values were calculated using the two-sided Mann–Whitney t-test except in (g, j, m) where the Wilcoxon matched-pairs signed rank test was used. Source data are provided as a Source Data file.