Knockdown of HDAC6 using siRNA and TRIM21 ubiquitin ligase in iBMDMs

For Hdac6-knockdown experiments using siRNA, iBMDMs were electroporated with two Hdac6 Silencer siRNAs (Thermo Fisher Scientific, catalog nos. 67164 and 158920 for siRNAs #1 and #2, respectively) or with Silencer siRNA Negative Control (Thermo Fisher Scientific, catalog no. AM4611). For HDAC6 protein knockdown using TRIM21 ubiquitin ligase, iBMDMs (3.0×10⁶) stably overexpressing mouse pSMPP-mCherry-TRIM21 (a gift from L. James, Addgene no. 104971) were electroporated either with 2 μg of rabbit anti-human HDAC6 IgG that cross-reacts with mouse HDAC6 (Cell Signaling Technology, catalog no. 7612) or with 2 μg of isotype control IgG (Agilent Dako, catalog no. X0903), respectively. Nucleofection of siRNAs and HDAC6 antibody was performed with the Nucleofector 2b device (Lonza, catalog no. AAB-1001) using the Y-001 program according to the manufacturer’s recommendations. siRNA- and HDAC6 antibody–electroporated cells were incubated for 72 and 24 hours, respectively, before being processed for further experiments.