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. 2020 Dec 23;218(2):e20201217. doi: 10.1084/jem.20201217

Figure 9.

Figure 9.

ZFP91 facilitates TCR-induced BECN1–PIK3C3 complex assembly. (A) Lysates from Zfp91+/+Foxp3-Cre and Zfp91fl/flFoxp3-Cre T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 0, 4, or 8 h were subjected to IP using an antibody against BECN1. (B) Quantifications of immunoprecipitated PIK3C3:PIK3C3 levels in Zfp91+/+Foxp3-Cre (WT) and Zfp91fl/flFoxp3-Cre (KO) T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 4 or 8 h are shown as the mean ± SEM of three experiments. (C) IB analysis of BECN1 ubiquitination using HEK293T cells transfected with the indicated expression vectors. (D) IB analysis of BECN1 K63–linked ubiquitination in Zfp91+/+Foxp3-Cre (WT) and Zfp91fl/flFoxp3-Cre (KO) T reg cells stimulated with antibodies against CD3 and CD28. (E) Quantifications of BECN1-K63-Ub:BECN1 levels in Zfp91+/+Foxp3-Cre (WT) and Zfp91fl/flFoxp3-Cre (KO) T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 0 or 4 h are shown as the mean ± SEM of three experiments. (F) Lysates from WT T reg cells stimulated with anti-CD3 and anti-CD28 antibodies were subjected to IP using an antibody against BECN1. Quantifications of immunoprecipitated ZFP91:ZFP91 levels are shown as the mean ± SEM of three experiments. (G) IB analysis of the indicated proteins in nuclear (NE) and cytoplasmic (CE) extracts of WT T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 0, 4, or 8 h. Quantifications of ZFP91:GAPDH levels and ZFP91:lamin B levels are shown as the mean ± SEM of three experiments. (H) IB and co-IP assays using the cytosol fractions of WT T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 0, 4, or 8 h. (I) Quantifications of immunoprecipitated ZFP91:BECN1 levels in the cytosol fractions of WT T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 0, 4, or 8 h are shown as the mean ± SEM of three experiments. (J) IB and co-IP assays using the cytosol fractions of WT T reg cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of LMB for 4 h. Ctrl, control. Quantifications of immunoprecipitated ZFP91:BECN1 levels are shown as the mean ± SEM of three experiments. (K and L) Lysates from WT T reg cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of LMB for 4 h were subjected to IP using an antibody against BECN1. Quantifications of immunoprecipitated ZFP91:ZFP91 levels (K) and immunoprecipitated PIK3C3:PIK3C3 levels (L) are shown as the mean ± SEM of three experiments. Data are representative of three independent experiments. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01. Two-tailed Student’s t test. MW, molecular weight.