SUMO1 protein, human

Samples were fixed, paraffin embedded, sectioned, and stained with hematoxylin/eosin for histological evaluation as described [57 (link)]. Tissue sections were subject to immunological staining with avidin:biotinylated enzyme complex as described [18 (link),58 (link)]. Proteins were extracted from TS cells using M-PER reagent (PIERCE) with the addition of protease inhibitor cocktail (Sigma-Aldrich), 1 mM sodium molybdate, 1 mM sodium vanadate, and 10 mM N-ethylmaleimide, or SDS lysis buffer (2% SDS, 10% glycerol, and 50 mM Tris, pH 6.8). Protein extracts were subject to immunoblotting as described [54 (link)]. Bound primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Vector Lab), followed by ECL-mediated visualization (GE HealthCare) and autoradiography. Mouse monoclonal antibodies anti-actin (Thermo Fisher; 1:1,000), anti-BrdU (Thermo Fisher; 1:300), anti-Cdx2 (BioGenex; 1:1), anti-MDM2 (Santa Cruz; 1:100), and anti-SUMO-1 (Zymed; 1:2,000); rabbit polyclonal antibodies anti-calnexin (Stressgene; 1:2,000), anti-cyclin D1 (Neomarker; 1:100), anti-Ki67 (Neomarker; 1:400), anti-laminin (Sigma-Aldrich; 1:25), anti-Myc tag (CalBioChem; 1:400), anti-Oct4 (Santa Cruz; 1:200), anti-p53 (Santa Cruz; 1:50), and anti-p450scc (Chemicon; 1:200); and goat polyclonal antibody anti-lamin B (Santa Cruz; 1:100) were used as primary antibodies. BrdU incorporation analysis was performed by intraperitoneal injection of BrdU (250 μg/g of body weight) into pregnant females for 1 h. Placentas were recovered, fixed, embedded, sectioned, and subject to immunostaining as described [18 (link),57 (link)].

Next, we aimed to find a machine‐learning method that would allow us to input the missing parts of the map. The first step toward this was to gather suitable features. We first evaluated the most promising features using linear regression and then applied a random forest model using all the available features.
The most important features were intrinsic, that is, directly derived from unused information in the screen. These are the average fitness across variants at the same position; the average fitness of multi‐mutant clones that contain the variant of interest; the estimated fitness according to a multiplicative model to infer mutant fitness A using a double mutant AB and single mutant B. Another set of features was computed from differences between various chemical properties of the wild‐type and mutant amino acids. These properties include size, volume, polarity, charge, and hydropathy. A third set of features is derived from the structural context of each amino acid position. This includes secondary structure, solvent accessibility, burial in interfaces with different interaction partners and involvement in hydrogen bonds or salt bridges with interaction partners. Secondary structures were calculated using Stride (Frishman & Argos, 1995). Solvent accessibility and interface burial were calculated using the GETAREA tool (Fraczkiewicz & Braun, 1998) on the following PDB entries: for UBE2I: 3UIP (Gareau et al, 2012); 4W5V (Boucher et al unpublished); 3KYD (Olsen et al, 2010); 2UYZ (Knipscheer et al, 2007); 4Y1L (Alontaga et al, 2015); for SUMO1: 2G4D (Xu et al, 2006); 2IO2 (Reverter & Lima, 2006); 3KYD (Olsen et al, 2010); 3UIP (Gareau et al, 2012); 2ASQ (Song et al, 2005); 4WJO (Cappadocia et al, 2015); 4WJQ (Cappadocia et al, 2015); 1WYW (Baba et al, 2005); for calmodulin: 3G43 (Fallon et al, 2009); 4DJC (Sarhan et al, 2012); and for TPK1: 3S4Y (Baker et al, 2001).
Hydrogen bond and salt bridge candidates were predicted using OpenPyMol and evaluated for validity by manual inspection. Additional features used are the BLOSUM score for a given amino acid change, the PROVEAN score, and the evolutionary conservation of the amino acid position. Conservation was obtained by generating a multiple alignment of direct functional orthologs across many eukaryotic species using CLUSTAL (Sievers & Higgins, 2014), which was used as input for AMAS (Livingstone & Barton, 1993). We then applied the complete set of features in a random forest model using the R package Random Forest (Breiman, 2001) version 4.6–12 with the default settings for all hyperparameters (n_tree = 500, m_try = n_feat/3, replace = TRUE, sampsize = n_obs, nodesize = 5, maxnodes = NULL, nPerm = 1). These procedures were implemented as part of a larger DMS analysis package (see “Code and data availability” section).

pSG5-BMLF1, pSG5-BMRF1, and pSG5-Rta have been described previously (58 (link)) and were obtained from Shannon Kenney (University of Wisconsin—Madison, USA); pCMV5.1/FLAG-BALF5, pCDNA-HA2-BALF2, pCDNA-HA2-BBLF2/3, pCDNA-HA2-BBLF4, and pCDNA-HA2-BSLF1 were obtained from Ya-Fang Chiu (Chang-Gung University, Taoyuan, Taiwan). pCDNA3-HA(2x)-BVLF1 and pCDNA3-HA-BcRF1 were obtained from Eric Johannsen (University of Wisconsin—Madison, USA) and have been previously described (27 (link)). Plasmids p509, encoding pCMV-BZLF1 (Zta), and p526, encoding oriLyt, have been described previously (59 (link)). p3803, a retroviral vector encoding eGFP-H2B, was described previously (13 (link)). p3622 is a version of p3803 encoding mCherry-H2B. The wild-type EBV plasmid p2089 has been described previously (60 (link)). pCDNA-HA3-BGLF4 was cloned by PCR amplifying BGLF4 from p2089 using primers 5′-CGCGGGTACCGCCACCATGTATCCATATGACGTTCCAGATTACGCTGATGTGAATATGGCTGCGGAG-3′ (forward) and 5′-CGCGGAATTCTCATCCACGTCGGCCATCTGGACC-3′ (reverse) and cloning it into pCDNA3-HA-SUMO1 plasmid (Addgene plasmid no. 21154) at EcoRI/Acc65I sites.

After validating RNA purity and quality, cDNA was subsequently synthesized, and RT-PCR was performed using a commonly used housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a positive control. Synthesis of cDNA was performed using an Affinity Script QPCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA) with a thermal cycler (GeneAmp PCR System 9700, Applied Biosystems, Waltham, MA, USA) under conditions of 25 °C for 5 min, 42 °C for 5 min, 55 °C for 40 min, and 95 °C for 5 min. In addition, cDNA was amplified in solutions containing EmeraldAmp PCR Master Mix (Takara, Tokyo, Japan) and the relevant primers. The 3′-UTR gene-specific primers were targeted against the genes listed in Table 1. Sequences for MKP-1, CEBPD, KLF4, LEF1, SUMO1/SENP5, MMP-8, KSR1, and NGFR were obtained from the National Center for Biotechnology Information (NCBI) database. Thermal cycler parameters for PCR were as follows. After initial denaturation at 97 °C for 5 min, 25–35 cycles of 95 °C for 45 s, 55–65 °C for 45 s, and 72 °C for 1 min were performed. A final elongation step was performed at 72 °C for 10 min. PCR reaction solutions were then loaded onto a 1.6% agarose gel. Electrophoresis (100 V, 31 min or 85 V, 39 min) was performed in 1× TAE buffer, using a Mupid-ex electrophoresis system (Advance, Tokyo, Japan). Gels were stained with ethidium bromide for 10 min. Bands were then visualized using a ChemiDoc XRS Plus imaging system and quantified using Image Lab Software, version 3.0 (Bio-Rad). The intensity of all samples obtained was expressed relative to GAPDH expression in each sample. All assays were performed in duplicate, and the mean value was used as the value for one individual. An experimental series was performed twice using samples from different rats. When the second series was assayed, several samples measured in the first series were included to correct the absolute counts. Such measurements were repeated 3 times to ensure reproducibility. In preliminary experiments, we first clarified the PCR cycle number at which the expression level of each gene saturates. Based on these results, we adopted the PCR cycle number lower than the gene expression level saturating in the present study. We also performed RT-PCR and electrophoresis in a dilution series of the amount of cDNA in a sample containing a high amount of each gene and confirmed that the relationship between the amount of cDNA and its OD was linear. The intensities obtained from each sample were within the linear range in the preliminary experiments described above.

The cells were lysed with the appropriate volume of Pierce™ IP Lysis Buffer (Thermo Fisher Scientific) supplemented with 1X PIC and 1X PMSF and 25 mM of N-ethylmaleimide (NEM) (Thermo Fisher Scientific). Lysates were placed on ice for 10 min and centrifuged at 4 °C for 15 min at 15,000× g to collect protein lysates. For immunoprecipitation, 500 µL of cell lysate (1 mg/mL) was diluted with the Pierce™ IP Lysis Buffer supplemented with PIC, PMSF, and NEM and incubated with PROX1 antibody (Cell Signaling Technology, Danvers, MA, USA) and as the control normal rabbit IgG (Cell Signaling Technology) overnight at 4 °C with rotation. After overnight incubation, 50 mL of protein A/G agarose beads were washed twice with the IP lysis buffer, were added to each sample, and incubated at room temperature for 2 h with gentle rotation, followed by elution with IgG Elution Buffer (ThermoScientific). As described above, the eluted samples were run in 12%, Bis-Tris, 1.0 mm, Mini Protein Gel. The PROX1 SUMOylation was detected by probing the blot with an anti-SUMO1 antibody (Cell Signaling Technology) [17 (link)].