Ha p62

PPAR transcriptional activity was monitored in vitro using a reporter construct consisting of three PPRE copies upstream of a luciferase reporter. At day 5 of differentiation, BAs differentiated from immortalized SVF were transiently transfected with the following plasmids using X-tremeGENE: PPREx3-TK-luc (Addgene#1015), pRL-TK (control Renilla), HA-p62, HA-NBR1 or Flag-PPARγ1 (Addgene#78769). The level of promoter activity was evaluated by determining the firefly luciferase activity relative to renilla luciferase activity using the Dual Luciferase Assay System (Promega) according to the manufacturer’s instruction.

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).