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. 2019 Oct 10;10(10):771. doi: 10.1038/s41419-019-2011-5

Fig. 5. Tethering autophagy adapters to subcellular structures does not trigger their removal.

Fig. 5

a Immunofluorescence staining for CALR in cell lines expressing ER–hook and SBP–GFP–LC3 (left) or p62-SBP–GFP (right) in the presence of biotin (40 µM), and after shifting to avidin (60 µM) for 12 h. Lower panels show same experiment but in the presence of 1 µM rapamycin (Rapa). Scale bar equals 10 µm. b As in (a), but for cell lines expressing Golgi hook, and staining for B4GALT1. c Quantification of relative fluorescence intensity (FI) of CALR immunofluorescence staining signal from images in (a). d As in (c), but of B4GALT1 immunofluorescence staining signal from images in (b). e Immunoblots of cell lines expressing ER hook and indicated SBP–GFP bait constructs in the presence of biotin (40 µM), after shifting to avidin (60 µM; 12 h), and after shifting to avidin + rapamycin (1 µM, 12 h). Primary antibodies used are indicated. f As in (e), but immunoblots of cell lines expressing Golgi hook. The two bands correspond to soluble and membrane-bound isoforms of B4GALT1. Immunoblots were quantified by densitometry and quantitative data are shown in (g) and (h). Bars indicate means ± standard deviation of at least three replicates (*p < 0.05 and **p < 0.01, two-tailed Student’s t test, compared to control cells)