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: Bio-protocol

Article Title: Expression and Analysis of Flow-regulated Ion Channels in Xenopus Oocytes

doi: 10.21769/BioProtoc.2224

Figure Lengend Snippet: A. Injection pipettes made of glass capillaries are used for injection of cRNAs encoding for ion channels and other proteins in Xenopus oocytes. Injection workstation components labeled as follows: 1. Stereo microscope with external light source; 2. Micromanipulator and magnetic holding device; 3. Nanoinjector; 4. Nanoinjector controller. B. Loading of injection needle with cRNA. The injection needle is filled with mineral oil. A drop of the solution containing the cRNA of interest is place in the center of a piece of Parafilm. cRNA solution is collect with the injection pipette using the ‘fill’ bottom in the nanoinjector controller. C. Close view of oocytes aligned in a Petri dish. To prevent rolling during injection, oocytes are placed in a Petri dish with a polypropylene mesh glued to the bottom.

Article Snippet: Harvesting of ovaries from female Xenopus laevis 2-L glass beaker Fine forceps for tissue collection (Roboz Surgical, catalog number: RS-5240) Fine scissors for tissue collection (Fine Science Tools, catalog number: 14060-09) Tissue forceps (Roboz Surgical, catalog number: RS-8162) B. Oocyte isolation and maintenance Dumont size 4 forceps (Fine Science Tools, catalog number: 11241-30) Clay Adams TM Nutator mixer (BD, catalog number: 421105) Oocytes transferring pipette: a polished glass Pasteur pipette (Fisher Scientific, Fisherbrand TM , catalog number: 13-678-20) Pipette pump (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F378980000) B.O.D. low temperature refrigerated incubator (VWR) C. Oocyte injection Programmable nanoliter injector (Drummond Scientific, model: Nanoject III) Steel base plate (WPI, catalog number: 5052) Magnetic holding stand (WPI, catalog number: M10) Three-axis manual micromanipulator (WPI, catalog number: M3301) Stereo microscope (Olympus, model: SZ61) Fiber optic illuminator and bifurcated light guide (WPI) Micropipette pullers (NARISHIGE, model: PC10) D. Two-electrode voltage clamp Steel base plate (WPI, catalog number: 5479) Steel base plate (WPI, catalog number: 5052) Magnetic holding devices, three (WPI, catalog number: M10) Three-axis manual micromanipulators, three (WPI, catalog number: M3301) Two-electrode voltage clamp amplifier (Molecular Devices, Axon Accessories, model: GeneClamp 500B) Headstages (Molecular Devices, Axon Accessories, model: HS-2A) Virtual ground bath clamp (Molecular Devices, Axon Accessories, model: VG-2A) Pipette holders (Molecular Devices, model: HL-U) Ag/AgCl pellets (2 mm diameter, 4 mm long) (WPI, catalog number: EP2) Digitizer (Molecular Devices, model: Digidata 1550B) BNC cables (A-M systems) Perfusion system (Warner Instruments, model: VC-6) Flow valve (Warner Instruments, model: FR-50) Vacuum attached waste bottle (Fisher Scientific, Fisherbrand TM , catalog number: FB3002000) Oocyte chamber (20 mm diameter and 6 mm deep ring with glued glass bottom) Silver wire AWG 26 for electrodes (WPI, catalog number: AGW1510) PC computer with Windows operating system

Techniques: Injection, Labeling, Microscopy, Transferring