Journal: PloS one

Article Title: Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion.

doi: 10.1371/journal.pone.0005780

Figure Lengend Snippet: Figure 1. Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (PlaH) and light chain (PlaL) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS- PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 mg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37uC prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 mg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 mg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (161010) from log phase cultures (JEC21) were incubated 4 hr at 37uC in the presence (lane 1) or absence (lane 3) of 50 mg plasminogen and separated into cell wall and cytosol components, as described in Methods. Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 mg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37uC with 50 mg plasminogen (lanes 1–4, respectively). doi:10.1371/journal.pone.0005780.g001

Article Snippet: Western Blot analysis: Electro-blotted PVDF membranes were first incubated overnight at 4uC in PBS with 1.5% BSA and 0.07% Tween 20 then exposed for 1 hr (25uC) to rabbit anti-human plasminogen (Fitzgerald Industries), diluted 1:1000 in PBS+1.5% BSA.

Techniques: Incubation, Saline, Labeling, Extraction, SDS Page, Western Blot, Binding Assay

Journal: PloS one

Article Title: Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion.

doi: 10.1371/journal.pone.0005780

Figure Lengend Snippet: Figure 6. Identification of plasminogen-binding cell wall proteins of C. neoformans by 2D-PAGE and LC-MS/MS. Two-dimensional gel electrophoretic characterization of the cell wall proteome from a silver-stained gel (A), and the corresponding plasminogen-binding proteins (B) by ligand (plasminogen) overlay and western blot analysis with anti-plasminogen antibody. Indicated are the positions of identified plasminogen- binding proteins. Identified proteins shown in (B) are listed in Table S2. (* indicates spots sequenced following excision from the PVDF membrane due to lack of detection by silver-staining). doi:10.1371/journal.pone.0005780.g006

Article Snippet: Western Blot analysis: Electro-blotted PVDF membranes were first incubated overnight at 4uC in PBS with 1.5% BSA and 0.07% Tween 20 then exposed for 1 hr (25uC) to rabbit anti-human plasminogen (Fitzgerald Industries), diluted 1:1000 in PBS+1.5% BSA.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Two-Dimensional Gel Electrophoresis, Staining, Western Blot, Membrane, Silver Staining

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