Gfp ub

The calcium channels used were rabbit Ca_V2.2 (GenBank Accession number D14157), rat Ca_Vα₂δ-1 (M86621), and rat Ca_Vβ1b (X61394), all expressed in the pMT2 vector. The green fluorescent protein mut3bGFP (GFP) (44 (link)) or mCherry was fused to the C terminus of Ca_Vβ1b. GFP-Ub and GFP-Ub^KO (18 (link)) were obtained from Addgene. The first intracellular loop of Ca_V2.2 (amino acids 356–483, beginning ESGEF … and ending … KAQ) had a palmitoylation sequence, MTLESIMACCL, added to the N terminus and an HA tag (TSYPYDVPDYA) added to the C terminus to give a construct termed palm Ca_V2.2 I-II-HA in this study. Further constructs were made where the HA tag was substituted with GFP or mCherry. The mutant palm Ca_V2.2 I-II K-R-HA was made by mutating all 12 lysine residues in the I-II loop to arginines, and two further constructs were made by mutating either the first four (palm Ca_V2.2 I-II 5′ K-R-HA) or the last eight (palm Ca_V2.2 I-II 3′ K-R-HA) lysines to arginines. The I-II loop containing the 12 lysine-to-arginine substitutions was inserted into the full-length Ca_V2.2, which also contained an extracellular HA tag (6 (link)) and GFP fused to the N terminus to give GFP-Ca_V2.2 K-R-HA. The sequences of all constructs were confirmed by DNA sequencing.

BAG3 deletion mutants were amplified by PCR from human BAG3 complementary DNA. Amplicons were cloned in the mammalian shuttle vector pShuttle-CMV (Agilent Technologies) downstream of the CMV promoter. For expression analysis of the mutant protein, a FLAG-tag (DYKDDDDK) was introduced at the N-terminal site. Expression of mutant protein was verified in HEK293 cells by Lipofectamine (Thermo Fisher) transfection. The plasmids mCherry-lamin B1-10 and mCherry-lamin A-C-18 were a kind gift from Michael Davidson lab (Addgene plasmid #55069), as were HA-p62 from Qing Zhong lab (Addgene plasmid #28027), and GFP-Ub from Nico Dantuma (Addgene #11928).

Cells were seeded on coverslips and cultured in 24-well plates for 24 h, followed by transfection with GFP-UB (11928) and RFP-LC3 (21075) (Addgene, USA) or RFP-mito and GFP-LC3 (GeneChem, Biotechnology, Shanghai, China) plasmids using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer's instructions. 48 h after transfection, cells were treated with different drugs for the indicated amounts of time, and then fixed with 4% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 10% FBS for 30 min. Cells were incubated with various primary antibodies at 4°C overnight, followed by incubation with the following secondary antibodies at 37°C for 1 h, as appropriate: Alexa Fluor 488 goat anti-mouse (Molecular Probes, A11001), Alexa Fluor 405 goat anti-mouse (Molecular Probes, A-31553), or Alexa Fluor 647 donkey anti-rabbit (Molecular Probes, A31573). Cells were visualized using a laser-scanning confocal microscope (LSM780NLO, Zeiss, Germany) or a confocal microscope with a live cell imaging chamber (DMI 6000B, Leica, Germany). Mitochondrial length and percentage of cells in which mitophagy occurred were determined blindly using randomized filenames (Filename randomizer, CodeUnit, Craig Lotter) and Zeiss LSM Image Examiner software.