Next, the average brightness of an empty portion of each picture was measured (background fluorescence) and subtracted from the maximal fluorescence for each bead [16]. 2.10. of Cx43 to LC3B and GABARAP. Overall, our findings describe an alternative mechanism whereby Cxs interact with LC3/GABARAP proteins, envisioning a new model for the autophagy degradation of connexins. and 32 C, cells were plated and monitored for the expression of GFP. Lentiviral vectors containing AS1842856 shRNA targeting ATG7 and the control empty vector were kindly provided by Dr. A.M. Cuervo (Albert Einstein College of Medicine, Yeshiva University, New York, USA). 2.5. siRNA-Mediated Knockdown siRNA targeting p62 (s16960 or s16961) and a non-targeting control AS1842856 sequence were obtained from Ambion. Cells were transfected with 20 nM siRNA using Lipofectamine 2000 (Grand Island, NY, Invitrogen) according to manufacturers recommendations. p62 knockdown was achieved after 24 h of transfection. 2.6. Immunoprecipitation and Western Blotting Cells were rinsed with phosphate buffered saline (PBS) at 4 C, resuspended in lysis buffer (190 mM NaCl, 50 mM Tris-HCl, 6 mM EDTA, 1% Triton X-100, pH 8.3) supplemented with protease inhibitor cocktail (Roche), 2 mM PMSF, 10 mM iodoacetamide, and incubated on ice for 10 min. The samples were then centrifuged at 10,000 for 10 min and the supernatants used for immunoprecipitation. Briefly, protein G was incubated with AS1842856 goat polyclonal antibodies directed against Cx43 or V5. Incubations proceeded for 1 h at 4 C, followed by incubation with supernatants for 3 h at 4 C. The samples were then centrifuged and the protein G-sepharose sediments washed 3 times in an appropriate washing buffer (500 mM NaCl, 50 mM Tris-HCl, 6 mM EDTA, 1% Triton X-100, pH 8.3), resuspended in Laemmli buffer and denatured at 70 C for 10 min. For Western blot analysis of the immunoprecipitated proteins, samples were separated using SDS-PAGE, transferred to a nitrocellulose membrane and probed with appropriate antibodies. Inputs represent about 10% of the total amount of protein in the lysates before immunoprecipitation. Immunoprecipitation controls were performed by pooling the lysates of two samples transfected and/or treated in the same conditions, separating them in two fractions and then proceeding with the immunoprecipitation without adding antibody to one of the samples (No Ab). The corresponding pooled lysate appears in the Western blot panels to the right of the No Ab samples. 2.7. Bacterial Protein Expression and Purification GST-Cx43WT_NT and GST-Cx43W4A+L7A_NT proteins were expressed in Escherichia coli BL21-CodonPlus (DE3)-RILP Cells (Agilent Technologies, Santa Clara, CA, USA). Bacteria were grown in Luria broth (LB) medium until OD600 0.8C1, induced with 0.1 mM isopropylthiogalactoside (IPTG) and grown at 37 C for 4 h. GST constructs were isolated from harvested cells using Glutathione Sepharose 4B (GE Healthcare, Buckinghamshire, UK) according to manufacturers recommendations. GFP-LC3B and GFP-GABARAP proteins were expressed in Escherichia coli Rosetta (DE3) pLysS cells. Cells were induced at an OD600 of 0.5 for 16 h at 18 C with 0.1 mM IPTG. Harvested cells were resuspended in lysis buffer 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.5, 500 mM NaCl, 10 mM imidazole, 2 mM MgCl2, 2 mM -mercaptoethanol, complete protease inhibitor (Roche, Basel, Switzerland) and DNase I and lysed using a freezeCthaw cycle followed by brief 30 s sonication. Lysates were cleared using ultracentrifugation at 140,000 g for 30 min at 4 C (Beckman, Brea, CA, USA, Ti45 rotor). Supernatants were applied to Ni-NTA columns (GE Healthcare, Buckinghamshire, UK), and constructs were eluted via a stepwise imidazole gradient Mouse monoclonal antibody to ATIC. This gene encodes a bifunctional protein that catalyzes the last two steps of the de novo purinebiosynthetic pathway. The N-terminal domain has phosphoribosylaminoimidazolecarboxamideformyltransferase activity, and the C-terminal domain has IMP cyclohydrolase activity. Amutation in this gene results in AICA-ribosiduria (50, 75, 100, 150, 200, and 300 mM) [16]. 2.8. AS1842856 GFP-Trap Pull-Down Assay Five microlitres of GFP-Trap beads slurry (ChromoTek, Munich, Germany) were mixed with a 5 M solution of GFP-fused bait proteins (GFP-LC3B or GFP-GABARAP) and incubated on a rotating wheel at 4 C for 1 h. Subsequently the beads were washed twice with 150 mM NaCl, 50 mM Tris at pH 7.4, and incubated with 20 g of prey solution (GST-Cx43WT_NT or GST-Cx43W4A+L7A_NT). Precipitates were analysed using Western AS1842856 blot using goat polyclonal antibodies against GST. 2.9. Microscopy-Based Protein-Protein Interaction Assay Five microlitres of glutathione Sepharose 4B beads slurry (GE Healthcare, Buckinghamshire, UK) were mixed with 20 g of GST-fused bait.