Fig. 5: ARRB2 negatively regulates autophagy induced by TLR3 and TLR4. | Cell Death & Disease

Fig. 5: ARRB2 negatively regulates autophagy induced by TLR3 and TLR4.

From: β‐arrestin 2 negatively regulates lung cancer progression by inhibiting the TRAF6 signaling axis for NF-κB activation and autophagy induced by TLR3 and TLR4

Fig. 5

A ARRB2-knockout (ARRB2KO) H1299 cells were generated by CRISPR/Cas9 gene-editing method as described in Material and Methods. ARRB2 expression was verified by anti-ARRB2 antibody, along with anti-GAPDH as a loading control. B, C Control (Ctrl) H1299 and ARRB2KO H1299 cells were treated with or without LPS (15 μg/ml) for 3 h. Endogenous immunoprecipitation (IP) assay was performed with anti-TRAF6 (B) or anti-BECN1 (C). IP complexes were immune-probed with anti-TRAF6, anti-BECN1, or anti-Ub antibody. D Ctrl H1299 and ARRB2KO H1299 cells were treated with vehicle (DMSO, 0.1% v/v concentration), PoIy I:C (25 μg/ml), LPS (15 μg/ml) for 6 h. Cell lysates were immunoblotted with an anti-LC3A/B antibody and anti-GAPDH (as a loading control). E Ctrl H1299 and ARRB2KO H1299 cells were treated with vehicle (DMSO, 0.1% v/v concentration), Poly I:C (5 μg/ml), or LPS (15 μg/ml) in the presence or absence of 3-MA (5 mM) for 6 h, and immunolabeled with LC3 antibody, as described in Material and Methods. Images shown are representative fluorescence confocal microscopic photographs. Quantification of the percentage of cells with autophagosomes is shown (±SD, n = 50 cells). Scale bar: 10 μm. F A schematic model of how ARRB2 interrupts the association between TRAF6 and BECN1 and inhibits autophagy through inhibition of BECN1 ubiquitination.

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