Poptn egfp

pN3-3xFlag-Control (Addgene, 107717, Guntram Suske Lab [75 (link)]); pmRFP-LC3 (Addgene, 21075, Tamotsu Yoshimori Lab [43 (link)]); pMXs-puro GFP-Sqstm1/p62 (Addgene, 38277, Noboru Mizushima Lab [49 (link)]); pBABE-puro mCherry-EGFP-LC3B (Addgene, 22418, Jayanta Debnath Lab [76 (link)]); pBABE-EGFP (Addgene, 36999, Debu Chakravarti Lab [77 (link)]); ECFP-SSH1ΔC (N461) (Dr. Mizuno lab [51 (link)]); EGFP-SSH1 (Dr. Storz lab [69 (link)]); mKeima-Red-Mito-7 (Addgene, 56018); pOPTN-EGFP (Addgene, 27052Beatrice Yue Lab [78 (link)]); pMXs-puro GFP-Sqstm1/p62ΔC (Addgene, 38282, Noboru Mizushima Lab [49 (link)]); pMXs-puro GFP-Sqstm1/p62 D337, 338, 339A (GFP-SQSTM1-LIR) (Addgene, 38280, Noboru Mizushima Lab [49 (link)]); HA-Ubiquitin (Addgene, 18712, Edward Yeh Lab [79 (link)]); pDR125 (Addgene, 37150, Dale Ramsden Lab); pN3-3xFlag-SSH1, mCherry-EGFP-Sqstm1/p62, mCherry-EGFP-Sqstm1^S403A, mCherry-EGFP-Sqstm1^S403E, pN3-Flag-SSH1ΔC, pN3-Flag-SSH1ΔN and pN3-Flag-SSH1ΔN^C393S were generated in this work (see methods below).

pOPTN-eGFP (#27052), pcDNA3-HA2-Keap1 (#21556), and pHA-tag (#55182) were purchased from addgene.org. For expressed co-IP assay, HEK293T cells were seeded in six-well plates until 80% confluency and transfected with indicated plasmids at a concentration of 2000 ng per plasmid per well by Lipofectamine 2000 according to the manufacturer’s protocol. Transfected cells were lysed by NP-40 buffer and split into two parts—200μl fractions for input assay and an 800 μl of fractions for co-IP. The input lysates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and analyzed by immunoblot. The co-IP lysates were incubated with 20 μl of EZview Red Anti-HA Affinity Gel (E6779-1ML, MilliporeSigma) for 24 hours. For endogenous co-IP assay, osteoclasts from 2-month-old Optn^+/+ mice were used. The co-IP lysates were precipitated with anti-KEAP1 antibody (sc-514914, Santa Cruz Biotechnology) or normal mouse immunoglobulin G control (#02-6502, Invitrogen) bound to protein G and protein A agarose beads (IP05, MilliporeSigma), respectively, for 24 hours. After the incubation, samples were centrifuged at 5000g for 1 min, and pellets were washed five times with NP-40 buffer. The proteins were lastly resolved on SDS-PAGE gels and analyzed by immunoblot.