Figure 1.

Efficient invalidation of autophagy in B cells from Atg5^f/− CD21 cre mice and Atg5^f/− Mb1 cre mice. Atg5^f/+ CD21 cre, Atg5^f/− CD21 cre, Atg5^f/− Mb1 cre, and Atg5^f/+ Mb1 cre were generated. B or T cells were purified from the spleen and cultured in vitro in the presence of different stimuli. Lysosomal protease inhibitors pepstatin A and E64d were added (+) or not (−) in the indicated conditions. (a) T cells from wild-type C57BL/6 mice (B6), Atg5^f/+ CD21 cre, or Atg5^f/− CD21 cre were left unstimulated (non-stim) or stimulated by anti-CD3 Ab for 18 h. Cells were lysed and immunoblots were performed against ATG5, LC3, and ACTB. (b and c) B cells from wild-type B6, Atg5^f/+ CD21 cre or Atg5^f/− CD21 cre (b), Atg5^f/− Mb1 cre or Atg5^f/+ Mb1 cre (c) were left unstimulated (non-stim) or stimulated by anti-IgM Ab for 18 h. Cells were lysed and immunoblots were performed against ATG5, LC3, and ACTB. (d) Upper panel. Quantification of Atg5 transcripts in B cells by real-time PCR relative to Gapdh expression. One sample from B6 mouse was used for each plate for normalization and its value is arbitrarily set to 1. Middle and lower panels. Densitometric analysis of ATG5 and LC3-II expressions relative to ACTB. The values corresponding to the non-stimulated condition in the presence of protease inhibitors were collected, and normalized to the ratios obtained from B6 wild-type mice on each blot to normalize results. The histograms represent the means with S.E.M. obtained for at least three and seven different blots, respectively, for ATG5 and LC3-II quantification. ns, non-significant; ***P<0.001 (Mann–Whitney U-test)