Fig 2: T lymphocytes express two major Dlg1 protein variants due to alternative splicing: Dlg1AB and Dlg1B.(A) Schematic of the analyzed areas of splicing in dlg1. (B) RT-PCR of cDNA from different murine T cells (CD8 = purified unstimulated primary OT-1 CD8+ T cells; CTL = purified OT-1 CD8+ T cell stimulated ex vivo with CD3/CD28 and expanded in rIL-2; OT-1H = CD8+ OT-1 hybridoma T cell line; BI-141 T hybridoma cell) using primers that flank the i1A/i1B (top) or i2/i3/i4/i5 (middle) splice region. Primers that lie within L27ß were also used (bottom). (C) Schematic of four possible dlg1 transcripts expressed in T cells. (D) BI-141 T cells infected with viruses encoding different Dlg1 splice variants were analyzed via western blotting for Dlg1 expression; Lck was used as a loading control. (E) Schematic representation of Dlg1 variants targeted for knockdown using miR-based knockdown viruses. (F) BI-141 T cells infected with knockdown viruses targeting L27ß, i1A, i1B or i3 were analyzed via western blotting for Dlg1; Lck was used as a loading control. (G) Schematic representation of the two major Dlg1 protein variants expressed in T cells.

Fig 3: The IKRLL motif mediates the inhibitory function of the helix.(A) CD4 signal normalized as a percent of the total is shown for each sucrose gradient fraction (left) along with the AUC analysis for the DRM (center) and DSM (right) fractions. (B) Cholera toxin subunit B (CTxB) signal normalized to CD4 signal detected is shown for each sucrose fraction (left) along with the AUC analysis for the DRM (center) and DSM fractions (right). (C) Lck signal normalized to CD4 signal is shown for each sucrose fractions (left) along with the AUC analysis for the DRM (center) and DSM (right) fractions. (D) IL-2 dose response to MCC peptide (left). AUC analysis as a measure of the response magnitude (center), and the average response to a low dose (41nM) of MCC as a measure of sensitivity (right) are shown. For (A–D), the data are presented as in Figure 2. Key: AUC = Area Under the Curve; DRM = Detergent Resistant Membrane; DSM = Detergent Soluble Membrane; CTxB = Cholera Toxin subunit B.

Fig 4: The GGXXG + CV+C motifs influence CD4 membrane domain localization and function.(A) CD4 signal for each sucrose gradient fraction is shown as a percent of the total CD4 signal detected in all fractions (left). The area under the curve (AUC) is presented for the normalized CD4 signal in the detergent resistant membrane (DRM) fractions (center) and detergent soluble membrane (DSM) fractions (right). (B) Cholera toxin subunit B (CTxB) signal is shown for each sucrose fraction normalized to the CD4 signal detected in the corresponding fraction (left). The AUC is shown for the normalized CTxB signal in the DRM (center) and DSM (right) fractions. (C) Lck signal is shown for each sucrose fraction normalized to the CD4 signal detected in the corresponding fraction (left). The AUC is shown for the normalized Lck signal in the DRM (center) and DSM (right) fractions. (D) IL-2 production is shown in response to a titration of MCC peptide (left). AUC analysis for the dose response is shown as a measure of the response magnitude (center). The average response to a low dose (41nM) of peptide is shown as a measure of sensitivity (right).For (A-C) each data point represents the mean ± SEM for the same three independent experiments (biological replicates). For (D), the dose response represents one of three experiments showing the mean ± SEM of triplicate wells (technical replicates). the magnitude and sensitivity data represents the mean ± of three independent experiments (biological replicates). One-way ANOVA with a Dunnet's posttest for comparisons with WT samples, or a Sidak's posttest for comparisons between selected samples, were performed. Key: AUC = Area Under the Curve; DRM = Detergent Resistant Membrane; DSM = Detergent Soluble Membrane; CTxB = Cholera Toxin subunit B.

Fig 5: Hex and Gal glycohydrolases increase their targeting to lipid microdomains after cell stimulation. (A) Gene expression analysis by Q-PCR of HEXB, HEXA, and GLB1 genes in resting and PHA-stimulated Jurkat cells. The ACTB gene was used as the endogenous control. The values are expressed as Relative Quantity (RQ). The mean ± SEM of three independent experiments is reported. *** p < 0.001 (PHA-stimulated vs. resting cells). Lipid microdomains were isolated from resting and PHA-stimulated Jurkat cells (1 × 108) by a discontinuous sucrose-density gradient. (B) Fractions were collected from the top to the bottom of the tube and were analyzed by immunoblotting for flot-2 and lck (#, p56lck; ##, p60lck). Representative Western blotting of five independent experiments is reported. (C) Distribution of Total Hex, Hex A, and Gal enzymatic activities is expressed as total mU (tot. mU) in each fraction. Values are the mean ± SEM of five independent experiments. *** p < 0.001 (PHA-stimulated vs. resting cells). LM, lipid microdomain fractions; H, high-density fractions; Rest, resting cells; PHA, PHA-stimulated cells.