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Fig. 2 | Journal of Biomedical Science

Fig. 2

From: Secretory autophagy promotes Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1

Fig. 2

Autophagy promotes TIMP1 secretion in a Rab37-dependent manner under starvation conditions. Lung cancer H460 cell line stably expressing Rab37 lentivirus shRNA was established. a Total protein of the H460 cells was extracted, and the protein levels of Rab37, LC3, and TIMP1 in cell lysate and in condition medium (CM) were collected for detection of specific cellular proteins as well as the secreted TIMP1 by immunoblotting using specific antibodies. β-Actin and the whole cell lysate with Coomassie blue staining were used as the internal controls. b H460 cells stably expressing lentivirus Rab37 or GFP shRNA were treated with amiodarone (2 μM) for 24 h. The protein level of TIMP1 in the conditional medium (CM), and the levels of Rab37, TIMP1, and LC3 protein in the cell lysate were evaluated by immunoblotting using specific antibodies. c OptiMEM is similar to DMEM and is specially used for Tat-D11 treatment. H460 cells were treated with optiMEM with 5% serum and different concentrations of Tat-D11 (0–20 μM) for 7 h, and the levels of LC3 protein were determined by immunoblotting using anti-LC3 antibody. β-Actin was used as the internal control. d H460 cells were treated with PBS, DMEM without 5% serum, optiMEM with 5% serum, and optiMEM + 5% serum + Tat-D11 (20 µM) for 7 h, and then condition media were collected, followed by ELISA detection to measure the level of TIMP1. In addition, active-form Rab37 (GTP-Rab37) in the total cell lysate of the four groups was pulled down by GTP-binding beads, followed by Rab37 antibody detection. **p < 0.01, ***p < 0.001, ns: not significant. Data were analyzed by Student’s t-test

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