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. 2022 Sep 5;11:e78383. doi: 10.7554/eLife.78383

Figure 8. CLS1 and CLS4 jointly modulate the ATG16L1-INPP5E interaction.

(a) The indicated EGFP-INPP5E constructs were coexpressed in HEK293T cells with Flag-ATG16L1, as shown. Lysates were immunoprecipitated with GFP-Trap beads and analyzed by Western blot with the indicated antibodies. (b) Quantitation of Flag-ATG16L1 co-IP by the indicated EGFP-INPP5E constructs in HEK293T cells. Co-IP levels, expressed as percentage of WT, are normalized by the amounts of both immunoprecipitated EGFP-INPP5Es and Flag-ATG16L1 lysate levels. Data are mean ± SEM of n=3 independent experiments and were analyzed by one-way ANOVA followed by Dunnett tests relative to WT. Significance is shown as p<0.01(**). (c) Schema of INPP5E structure depicting CLS1-4 and the proteins through which they regulate INPP5E ciliary targeting, as shown herein.

Figure 8—source data 1. Uncropped immunoblots from Figure 8a.
Relevant bands are inside rectangles. See Figure 8 for more details.
Figure 8—source data 2. Uncropped immunoblot from Figure 8a (IP: EGFP; WB: ATG16L1).
Figure 8—source data 3. Uncropped immunoblot from Figure 8a (IP: EGFP; WB: EGFP).
Figure 8—source data 4. Uncropped immunoblot from Figure 8a (Lysates, WB: ATG16L1).
Figure 8—source data 5. Source data from Figure 8b.
Data from n=3 experiments.

Figure 8.

Figure 8—figure supplement 1. INPP5E targeting to the T-cell immune synapse is CLS-independent.

Figure 8—figure supplement 1.

(a) Schema depicting an immune synapse (IS) between an antigen-presenting cell (APC) and a T-cell. Herein, Raji and Jurkat cells were used as APC and T-cells, respectively. F-actin is a marker well known to accumulate in productive immune synapses. (b) Jurkat cells were challenged with CMAC-labelled SEE-pulsed Raji cells to induce synaptic conjugate formation. Cells were fixed, permeabilized and stained with Alexa Fluor 546-conjugated phalloidin (F-actin, red) and anti-INPP5E antibody (green). CMAC (7-amino-4-chloromethylcoumarin) is shown in blue. Scale bar, 10 µm. (c) Jurkat cells expressing the indicated EGFP fusion proteins were challenged and stained as in (b), except that an anti-EGFP antibody (green) was used instead of anti-INPP5E. Scale bar, 10 µm. Cells in (b–c) were imaged by epifluorescence microscopy.