Skip to main content
. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Nature. 2021 Nov 18;600(7888):308–313. doi: 10.1038/s41586-021-04109-7

Figure 3. Sec31a protects Sec13 from Skp1-mediated proteasomal degradation.

Figure 3.

(a) Immunoblot analysis of indicated protein expression in sgNTC- or sgSec31a-transduced cells treated with MG132 or DMSO for 9 h. Lower, quantification of relative Sec13 expression (n = 3 samples each group). (b) Control (sgNTC)- or Sec31a-null HEK293T expressing HA-tagged WT or K260R mutant Sec13 were treated with cycloheximide (CHX) for indicated times. Immunoblot analysis of HA and Hsp90 and quantification of relative Sec13 abundance (n = 3 samples each group). (c) T cells expressing HA-tagged WT or K260R mutant Sec13 were stimulated with α-CD3/CD28 for 48 or 72 h (with MG132 treatment for the last 6 h), followed by immunoprecipitation with anti-HA antibody. Immunoblot analysis for HA and β-Actin. (d) Indicated sgRNA-transduced cells were mixed with sgNTC (mCherry+; ‘spike’)-transduced cells, and stimulated with TCR for 3 h. Quantification of relative p-S6 level (n = 3 samples each group). (e) Immunoblot analysis of indicated protein expression and quantification of relative Sec13 abundance in indicated sgRNA-transduced cells (n = 4 samples each group). (f) Quantification of relative proportion of donor-derived (CD45.1+) T cells in the spleen of LCMV-infected mice (CD45.2+) at 7 d post-infection. See also Extended Data Fig. 7k (n = 10 mice per group). Mean ± s.e.m. (a, b, d–f). NS, not significant; ***P < 0.001; one-way ANOVA (a, d–f); two-way ANOVA (b). Data are representative of two (b–d) or three (e), or pooled from two (f) or three (a) experiments.