Generation of TRPV4 and Ubiquitin Constructs

TRPV4-FLAG in pcDNA3.1 was previously described (103 (link)). TRPV4-V5-FLAG was generated using Gibson Assembly Master Mix (New England Biolabs), by inserting the V5 tag sequence into the middle position of the first TRPV4 extracellular loop. TRPV4-V5-EGFP was generated by replacing the FLAG tag of the TRPV4-V5-FLAG construct with EGFP cloned from pEGFP-C1 (Clontech), as previously described (32 (link)). TRPV4 K>R mutants (All-K-IDR; 5K-IDR; 5K-CTD; K130, 136R), N-terminally Myc-tagged ITCH (NP_001311127.1), and ITCH C830A (ITCH-CI) were synthesized by GenScript in the pCI-Neo vector. Myc-NEDD4 and Myc-NEDD4-CI were generated by replacing the N-terminal HA tag in pCI-HA-NEDD4-CI (Addgene; plasmid nos.: 27002 and 26999) with a Myc tag using Gibson Assembly Master Mix (New England Biolabs). The expression plasmids for PACSIN1-Myc, PACSIN2-Myc, and PACSIN3-Myc were generous gifts from Dr Markus Plomann (University of Cologne).
The following plasmids were obtained from Addgene: pRK5-HA-Ub-K0 (17603), pRK5-HA-Ubiquitin-WT (catalog no.: 17608), pLenti-puro-HA-Ubiquitin (catalog no.: 74218-LV), pCI-HA-NEDD4 (catalog no.: 27002), pCI-HA-NEDD4-DD (catalog no.: 26999), HA-Ubiquitin (catalog no.: 18712), and RhoA-Myc (catalog no.: 12962).