Ub k63

The full-length open reading frame (ORF) of LAD1 (NM_005558.4) was used to generate a PCR amplicon, which was then cloned into the HA-tagged pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro (PCDH) vector. Using the same strategy, MAEA (NM_001017405.3) and Vimentin (NM_003380.5) plasmids were cloned into pCDNA3.1 harboring either an MYC-tag or a Flag-tag. Plasmids of Ub-K63 and Ub-K48 with an HA-tag were purchased from Addgene. The shRNA of LAD1 was purchased from Genepharma (Shanghai, China). LAD1 target sequences included: ShRNA-1, 5-GCCTCAGAGAAGACATCTCTA-3; ShRNA-2, 5-CTTTCGGATGAAACCCAAGAAA-3. Lentivirus infection was used to generate stable cell lines, and the transient infection method was the same as in the previously reported study [23 (link)].

Transfection of HEK293 cells with siRNA was performed using Viromer (Lipocalyx, Halle, Germany) according to the manufacturer’s instructions. siRNA oligonucleotides were purchased from Sigma Aldrich (Sigma-Aldrich, Taufkirchen, Germany). The following siRNA sequences were used: siGSK-3β: EHU0794515, SIHK0873, SIHK0872, siGSK-3α: EHU040791, SIHK0869. The plasmids for GSK-3β have been described previously48 (link). The full-length cDNA of human NEMO was cloned into pTagGFP-N or pTagRFP-N (Evrogen). Site-directed mutagenesis of the NEMO (serine to alanine or aspartic acid) was performed using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA). Mutations were verified by DNA sequence analysis. Ubiquitin wild-type (Addgene plasmid 17606; Cambridge, UK) and mutated ubiquitin, Ub-K48 and Ub-K63 (Addgene plasmids 17605, 17608), were used and have been described elsewhere54 (link).