Journal: Cell reports

Article Title: Impact of WIN site inhibitor on the WDR5 interactome

doi: 10.1016/j.celrep.2020.108636

Figure Lengend Snippet: (A) Endogenous PDPK1 was recovered from lysates of the indicated cell lines and probed for co-precipitating WDR5 by IB. Inputs for PDPK1 are 10%–20%. Inputs for WDR5 are 1%–5%. n = 3 biological replicates. (B) Proximity ligation assay with FLAG and WDR5 antibodies in U2OS cells stably expressing FLAG-tagged PDPK1. Cells were treated overnight (30 μM C6/C6nc) before analysis; scale bar, 20 μm. n = 3 biological replicates. (C) HEK293 cells were treated overnight with 30 μM C6 or 5 μM GSK470, lysates prepared, and a PDPK1 IP performed. IB was then performed for the indicated proteins. Inputs are 5% for PDPK1 and 1% for all others. n = 3 biological replicates. (D) HEK293 cells were fractionated into cytosolic (S2), soluble nuclear (S3), and chromatin-associated (P3) fractions. Equal amounts of each fraction were analyzed by IB with the antibodies against the indicated proteins. H3 (nuclear) and α-tubulin (cytosolic) are controls for fractionation. n = 3 biological replicates. (E) Cytosolic and nuclear lysates from HEK293 cells were subject to IP with PDPK1 antibody or an IgG control and immunoblotted with antibodies against the indicated proteins. A short and long exposure of the WDR5 IB are shown. n = 3 biological replicates. (F) PDPK1 possesses two WIN-like motifs centered on R3 and R238. (G) FLAG-tagged PDPK1 (WT and the R3A and R238A mutants) were transiently expressed in HEK293 cells; lysates were prepared and subject to IP with anti-FLAG beads. Immune complexes were probed for PDPK1 or endogenous WDR5 by IB. n = 3 biological replicates. (H) FLAG-tagged PDPK1 (WT and the R3A) was transiently expressed in HEK293 cells, recovered by FLAG-IP, resolved by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were then incubated with recombinant WDR5 followed by anti-WDR5 antibody. n = 3 biological replicates. (I) In vitro -transcribed and -translated PDPK1-FLAG variants were incubated with recombinant 6xHis-SUMO-WDR5 proteins, recovered with Ni-NTA agarose, and analyzed by IB. n = 2 biological replicates. PH, pleckstrin homology domain. See also .

Article Snippet: PolyScreen PVDF Hybridization Transfer Membrane , PerkinElmer , Cat# NEF1002.

Techniques: Proximity Ligation Assay, Stable Transfection, Expressing, Fractionation, Control, SDS Page, Membrane, Incubation, Recombinant, In Vitro

Journal: Kidney360

Article Title: Mannose Binding Lectin Is Hydroxylated by Collagen Prolyl-4-hydroxylase and Inhibited by Some PHD Inhibitors

doi: 10.34067/KID.0000092020

Figure Lengend Snippet: Some PHD inhibitors inhibit MBL oligomerization. (A) Representative Western blot showing the effects of PHD inhibitors (roxadustat [Rox], molidustat [Mol], and vadadustat [Vad], all at 10 µM) on expression of MBL, P4HA1, HIF1α, and α-tubulin. (B) Secreted MBL in light fraction (F1–F5) or heavy fraction (F6–F10) separated by sucrose gradient after 24-hour treatment with respective PHD inhibitors. (C) Quantification of the percentage of secreted MBL in the heavy fraction (compared to total MBL) in samples treated with different PHD inhibitors is shown. **P<0.01, ***P<0.001. (D) Representative distribution of relative proportion of secreted MBL (normalized to total secreted MBL) in ten fractions separated by sucrose density gradient for one sample from each group. Uncropped blots are provided in Supplemental Figure 6.

Article Snippet: Dimethyloxaloylglycine (DMOG; catalog number A4506) and molidustat (catalog number BAY85-3934) were purchased from ApexBio Technology.

Techniques: Western Blot, Expressing

Journal: Kidney360

Article Title: Mannose Binding Lectin Is Hydroxylated by Collagen Prolyl-4-hydroxylase and Inhibited by Some PHD Inhibitors

doi: 10.34067/KID.0000092020

Figure Lengend Snippet: Prolyl hydroxylation in the collagen-like region is significantly inhibited by roxadustat and vadadustat but not molidustat. Cells were treated with 10 µM of roxadustat, vadadustat, and molidustat for 24 hours. (A) Representative Western blots showing the effects of 24 hours of treatment with different PHD inhibitors on various proteins of interest in the lysates of MBL-expressing HEK293S cells. (B) Representative Western blots showing the effects of 24 hours of treatment with different PHD inhibitors on MBL secretion in the supernatant. The supernatant from three biologic replicates was concentrated and mass spectrometry analysis on the samples without knowledge of the treatments was undertaken. (C) Relative area of prolyl hydroxylated peptides covering the collagen-like domain containing hydroxylation at various proline residues (normalized to total area of MBL peptides within the collagen-like domain) in different conditions is shown. (D) Relative area of lysyl-modified (open circles) and unmodified (open squares) peptides covering the collagen-like domain (normalized by total area of MBL peptides within the collagen-like domain) in different conditions is shown. **P<0.01, ***P<0.001. Uncropped blots are provided in the Supplemental Figure 8.

Article Snippet: Dimethyloxaloylglycine (DMOG; catalog number A4506) and molidustat (catalog number BAY85-3934) were purchased from ApexBio Technology.

Techniques: Western Blot, Expressing, Mass Spectrometry, Modification