Bluo gal

Under deep pentobarbital anesthesia, mice were perfused with PBS, followed by 2.2% formaldehyde and 0.2% glutaraldehyde in PBS. Brains were removed and fixed for 1 h in the same fixative at 4 °C and then soaked in 30% sucrose in 0.1 m phosphate buffer for 12 h at 4 °C. The brains were frozen in tissue-tek OCT compound and cut at 30 μm thickness. Sections were subjected to lacZ staining overnight at 37 °C. LacZ staining solution contains 0.5 mg ml⁻¹ Bluo Gal (Invitrogen, Carlsbad, CA, USA), 3 mm K₄Fe(CN)₆, 3 mm K₃Fe(CN)₆ and 1 mm MgCl₂ in PBS.

WT and catalytically reduced CSN^5H138A bacmids were a kind gift from Radoslav Enchev (The Francis Crick Institute, London)¹⁹ . pcDNA3-myc3-CUL2 was a gift from Yue Xiong (Addgene plasmid #19892^{35 (link)}). HA-VHL wt-pBabe-puro was a gift from William Kaelin (Addgene plasmid #19234^{36 (link)}). RBX1, ELOB and ELOC were cloned from cDNA from the Mammalian Gene Collection purchased from Dharmacon. To improve protein yield, an N-terminally truncated (1–53) natural isoform of VHL was also produced for use with MS. Both isoforms of VHL were subcloned into a pET-52b(+) vector (Novagen) to add an N-terminal Step-Tag II. Genes were assembled into pACEBac1 using I-CeuI/BstXI restriction sites via the MultiBac system^{37 (link)}. RBX1 and CUL2 were assembled into one vector and ELOB, Strep II-VHL(ΔN) and ELOC into a second vector. Correct assembly was confirmed by sequencing of entire genes. DH10EmBacY cells were transformed with each assembly and blue/white selection was performed on l-agar plates containing 50 μg ml⁻¹ kanamycin, 7 μg ml⁻¹ gentamycin, 10 μg ml⁻¹ tetracyclin, 100 μg ml⁻¹ Bluo-Gal (Thermo Scientific) and 40 mg ml⁻¹ IPTG. DH10MultiBac bacmid DNA was isolated from single white colonies. Recombinant baculoviruses were generated in Sf9 insect cells (a clonal isolate from Sf21, Life Technologies #11496015) using standard amplification procedures.

The sFlt sequence for the first 3 Ig-like extracellular domains of sFlt-1 was cloned as previously described [13 ] into the pFastBac1 plasmid (Life Technologies) and then transformed into DH10Bac E.coli, which were plated on triple antibiotic plates containing kanamycin (50 μg/mL, Sigma Aldrich), gentamicin (7 μg/mL, Sigma Aldrich), tetracycline (10 μg/mL, Sigma Aldrich), IPTG (40 μg/mL, Sigma Aldrich) and Bluo-gal (100 μg/mL, Thermo Fisher Scientific). The sFlt gene-containing bacmid was isolated from DH10Bac E.coli (Life Technologies) and transfected into SF9 insect cells for virus production (provided by the Tissue Culture Facility, UC Berkeley). Virus was then used to infect High Five insect cells (provided by the Tissue Culture Facility, UC Berkeley) to induce sFlt protein expression. After 3 days, protein was purified from the supernatant using Ni-NTA agarose beads (Qiagen Laboratories). Recombinant sFlt was eluted from the Ni-NTA beads using an imidazole (Sigma Aldrich) gradient and then concentrated and buffer exchanged with 10% glycerol/PBS using Amicon Ultra-15mL Centrifugal devices (EMD Millipore). The protein solution was sterile filtered and the concentration was determined by BCA assay (Thermo Fisher Scientific). The molecular weight of sFlt was determined from a 4–20% gradient SDS-PAGE gel and estimated to be 50 kDa [12 (link)].