Ha ubiquitin

Nine human E3 ligase ORFs and human YAP and SKP2 ORFs were individually subcloned into the pCDH-MYC vector. Sixty-eight human DUB ORFs were individually subcloned into the pBabe-SFB vector. The His-GST-OTUD1 (#61405), MYC-SKP2 (#19947), and HA-ubiquitin (wild-type: #17608; K48R: #17604; K48: #17605; K63: #17606) constructs were from Addgene. The SFB-YAP, MYC-YAP, and HA-YAP constructs were gifts of Dr. Junjie Chen. The YAP-TEAD luciferase reporter construct was from Dr. Mien-Chie Hung (MD Anderson Cancer Center, Houston, USA). The K63R mutant of HA-ubiquitin, the lysine-to-arginine mutants of SFB-YAP and MYC-YAP, the C320S and H431R mutants of SFB-OTUD1, and the C320S mutant of His-GST-OTUD1 were generated using the QuikChange® kit from Agilent Technologies and validated by sequencing. Xpress-tagged full-length SKP2 and deletion mutants were from Dr. Hui-Kuan Lin’s lab (Wake Forest School of Medicine, Winston-Salem, USA). The UBCH5c shRNA (clone ID: NM_003340.4-849s1c1) and OTUD1 shRNA (clone ID: NM_001145373.2-1401s21c1 and NM_001145373.2-1725s21c1) constructs were from Sigma. The SKP2 shRNA (clone ID: V2LHS_199794 and V2LHS_199552) and YAP shRNA (clone ID: V2LHS_65508) constructs were from Dharmacon. siRNAs (CUL1: EHU070971; SKP1: EHU134251; OTUD1: EHU126001) were from Sigma. The human RBX1 siRNA was synthesized by Sigma and the sequence is 5′-GACTTTCCCTGCTGTTACCTAA-3′.

The following expression vectors were used in the present study: p4xCSL-firefly luciferase (Addgene #41726), pCAGGS-NICD (Addgene #26891), DN-dynamin 1 (Dynamin 1 K44A, a gift from Dr. Sandra Schmid), DN-dynamin 2 (GFP-dynamin 2 K44A, Addgene #22301), HA-Ubiquitin (Addgene #18712), pHR_SFFV_LaG17_synNotch_TetRVP64 (Addgene #79128), pHR_EGFPligand (Addgene #79129), pCS2-Notch1 FL-6MT (Addgene #41728), pCMV-mouse Dll1 (OriGene), pCMV-mouse Dll4 (OriGene), p6872 pHAGE-N-V5-MAML1-FL (Addgene #37048), and pHes1-GFPd2 (Addgene #14808). pCS2-Notch1 L468A was constructed by site-directed mutagenesis using the PrimeSTAR Mutagenesis Basal Kit (Takara). DN-MAML1 lacking the NICD-binding domain (13L-74H) and Dll1ΔC and Dll4ΔC lacking the intracellular domain (Dll1:569V-728L, Dll4:552A-686V) were amplified by polymerase chain reaction (PCR) and subcloned into pCAG-RB using the In-Fusion HD Cloning Kit (Clontech). pCMV-HA-Notch1 and pCMV-Myc-Ubiquitin were constructed by subcloning the cDNAs of Notch1 or Ubiquitin into pCMV-HA-N or pCMV-Myc-N (Clontech), respectively. Mouse and chick Psd1 cDNAs (ENSMUSG00000023452 and ENSGALT00000011126.6, respectively) were chemically synthesized and subcloned into the pGL3-promoter or pCMV-Myc-N vector.

HEK293T cells (~ 5 × 10⁶) were transfected with Flag-IRF3, His-UBE2J1 (or vector), together with HA-ubiquitin, HA-K48 ubiquitin or HA-K63 ubiquitin plasmids (Addgene, USA), respectively. 48 h later, cells were treated with proteasome inhibitor MG132 (20 μM, Sigma-Aldrich, USA) for another 6 h before harvest.
Immunoprecipitation were performed with anti-Flag® M2 Affinity Gel (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Briefly, 500 μl of cell lysates were incubated with 100 μl of the anti-Flag M2 Affinity Gel overnight at 4 °C. The beads were washed four times with 1 ml of lysis buffer containing 500 mM NaCl. The immunoprecipitated Flag-IRF3 proteins were then eluted and subjected to western blots. The co-immunoprecipitated UBE2J1 and ubiquitination signal on Flag-IRF3 was detected using anti-His and anti-HA antibodies, respectively.

FLAG-, Myc-, GFP-, or monomeric red fluorescent protein (mRFP)-tagged PIP5Kα, FLAG- or GFP-tagged PIP5Kα kinase-dead (KD) mutant (D309N, R427Q), and the N-terminal (1–65 aa), catalytic (66–434 aa), CT (435–546 aa), and CT-deleted (1–434 aa; ΔCT) regions of PIP5Kα subcloned into pGEX-6P-1 and/or pcDNA3-FLAG vectors have been described previously [35 (link), 36 (link)]. HA-tagged PIP5Kα, PIP5Kβ, or PIP5Kγ90 expression plasmids were previously provided by Dr. Michael Krauss (Leibniz-Institute for Molecular Pharmacology, Berlin, Germany) [37 (link)]. FLAG- (#11623), HA- (#32836), or GFP-tagged (#84293) Merlin, glutathione S-transferase (GST, #11631)- or FLAG-tagged (#11625) Merlin FERM domain, and FLAG-YAP (#66853), HA-TAZ (#32839), FLAG-TAZ (#27318), HA-NEDD4 (#27002), HA-ubiquitin (#17608), and HA-ubiquitin K48R (#17604) were obtained from Addgene (Cambridge, MA, USA). The F1 (18–98 aa), F2 (111–213 aa), and F3 (221–312 aa) regions of the Merlin FERM domain [23 (link)] were amplified from the GFP-Merlin plasmid by polymerase chain reaction (PCR) and subcloned into the EcoRI–BamHI sites of pEGFP-C2 vector. Myc-Merlin and HA-LATS1 plasmids were provided by Dr. Eunjeong Seo (OliPass Corporation, Yongin, Gyeonggi, Republic of Korea) and HA-Merlin L64P was a gift from Prof. Jung Soon Mo (Ajou University, Suwon, Republic of Korea).

We constructed lentivirus to overexpress NFIC (Lv-NFIC) or GFP (Lv-GFP) as a control in 293 T cells and treated cells with 2 μg·mL⁻¹ puromycin to select. 293 T cells were then transfected with HA-Ubiquitin (17608; Addgene) and USP34 siRNA. Cells were treated with 10 μmol·L⁻¹ MG132 for 4 h before collection of cell lysates. The supernatant was co-immunoprecipitated with anti-NFIC antibody and protein A/G magnetic beads. Then the sample was subjected to Western blot analysis.