Journal: eLife

Article Title: Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics

doi: 10.7554/eLife.83477

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , AlphaScreen SureFire pERK 1/2 (Thr202/Tyr204) Assay Kits , PerkinElmer , , .

Techniques: Purification, Expressing, Plasmid Preparation, Recombinant, Dominant Negative Mutation, Amplified Luminescent Proximity Homogenous Assay, Software

Journal: Journal of biomolecular screening

Article Title: Multidimensional profiling of CSF1R screening hits and inhibitors: assessing cellular activity, target residence time, and selectivity in a higher throughput way.

doi: 10.1177/1087057111418113

Figure Lengend Snippet: Fig. 2. Cellular assays for colony-stimulating factor 1 receptor (CSF1R). (A) GW-2580 effects on M-NFS-60 and NS0 cell proliferation. (B) Sorafenib effects on CSF1R and extracellular signal-regulated kinase (ERK) phosphorylation after CSF1 stimulation of THP1 cells. (C) AZD6244 effects in the same assays as panel B. The large difference in EC50 reflects that AZD-6244 is an MEK inhibitor. (D) Top panel: Western blot of lysed THP1 cells with an anti-phosphoERK1/2 antibody (phosphoERK1/2 bands are indicated by labels ERK1 and ERK2). Lane 1: no stimulus. Lane 2: CSF1 stimulus inhibited by GW-2580. Lane 3: CSF1 stimulus only. Lane 4: empty. Lane 5: marker. Bottom panel: control for total ERK. The ERK1 band (not indicated) is poorly visible just above the ERK2 band. Lanes in the bottom blot were digitally shuffled to match the lanes in the top blot.

Article Snippet: CSF1R and ERK target engagement assays Human monocytic tHp1 cells (atcc no. tiB-202) were stimulated with csf1, after which csf1r autophosphorylation and erk1/2 kinase activation were measured. tHp1 cells were cultured in Dulbecco’s modified eagle’s medium (Dmem)/f-12 medium (without phenol red) and supplemented with l-glutamine, penicillin, streptomycin, and 10% heat-inactivated fBs. in each well of a 96-well culture plate, 250 000 cells were dispensed (at 2·106 cells/ml) and rested overnight at 7 °c and 6% co2. the next day, compound was added to a final Dmso concentration of 0.1%, followed by 30 min preincubation and stimulation with 500 ng/ml of human csf1 for 5 min, after which the cells were put on ice. lysis buffer was added (from the perkinelmer [waltham, ma] alphascreen p-erk surefire kit), and cells were lysed for 10 min on a shaker. the lysates were frozen at –80 °c for further use. for detection of csf1r phosphorylation, a human phospho-csf1r duoset kit was used (Dyc3268; r&D systems, minneapolis, mn). maxisorb 96-well plates (nunc, roskilde, Denmark) were coated with mouse antihuman csf1r, blocked, and incubated with 100 μl of tHp1 cell lysate for 2 h at room temperature. after washing, the plates were incubated with horseradish peroxidase (Hrp)–labeled anti-ptyr. signal was detected by incubating with chromogen for 20 min and then reading absorbance at 450 nm on an enVision plate reader (perkinelmer). for detection of phosphorylated erk1/2 levels, 12.5 μl lysate was thawed out and mixed in a 384-well optiplate (perkinelmer) with 12.5 μl of surefire perk1/2 (cat. no. tgres10k; perkinelmer) bead mix that was prepared according to the manufacturer’s instructions. the mixture was shaken and placed in the dark for 2 h at room temperature. the fluorescent signal, which results from the proximity of two labeled antibodies against erk1/2 and phoshorylated erk1/2(pthr202/ tyr204) and diffusion of activated oxygen between them, was quantified in an enVision plate reader. for checking specificity of the signal, we ran western blots in a standard fashion. for primary antibodies, we used antiphosphorylated erk1/2 (cat. no. 4377; cell signaling, Danvers, ma) or anti-erk1/2 (cat. no. 700012; life technologies); for secondary antibody, we used goat-anti-rabbit Hrp (cat. no. w4011; promega, madison, wi). signal was generated using ecl mix (pierce, rockford, il) and captured on a gel imaging system (Fig. 2).

Techniques: Phospho-proteomics, Western Blot, Marker, Control