Purification of Apg2, Hsc70, and DNAJB1

The cDNAs of Apg2 (HSPH2), Hsc70 (HSPA8), and DNAJB1 were obtained from Addgene and cloned into pE-SUMO vector (LifeSensors, Malvern, USA). The mutants Apg2ΔAS and Apg2ΔC, carrying a deletion from residue 504 to 569 and 702 to 840, respectively, were cloned by fusing two PCR fragments corresponding to the upstream and downstream sequences of these protein segments [88 (link)] and verified by sequencing. Recombinant proteins containing a tag with 6 histidines and SUMO fused to the N-terminus were expressed in BL21 Rosetta cells and purified with a first step of affinity chromatography using NiNTA (Qiagen, Hilden, Germany) columns, followed by treatment with His-Ulp1 to cleave the tag, and a final NiNTA column in which the pure protein eluted in the unbound fraction [89 (link)].